IgG Fc Variants for Veterinary Use

ABSTRACT

Provided are various embodiments relating to variant IgG Fc polypeptides of companion animals having increased Protein A binding for ease of purification, decreased C1q binding for reduced complement-mediated immune responses, decreased CD16 binding (e.g., for reduced antibody-dependent cellular cytotoxicity (ADCC) induction, increased stability, and/or the ability to form heterodimeric proteins. In addition, various embodiments relating to antibodies and fusion proteins comprising such variant IgG Fc polypeptides are provided. Also provided are various embodiments relating to contiguous polypeptides comprising one or more variant GLP1 polypeptide(s) having improved serum half-life. Further provided are various embodiments relating to contiguous polypeptides or heterodimeric polypeptides comprising a GLP1 polypeptide and a glucagon polypeptide as a dual GLP1 receptor and glucagon receptor agonist. In various embodiments, such polypeptides may be used to treat, for example, diabetes, obesity, or related indications, in companion animals, such as canines, felines, and equines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/545,858, filed Aug. 15, 2017, which is incorporatedby reference herein in its entirety for any purpose.

FIELD

The present disclosure relates to variant IgG Fc polypeptides ofcompanion animals with enhanced features, including increased Protein Abinding (e.g., for ease of purification), decreased C1q binding (e.g.,for reduced complement-mediated immune responses), decreased CD16binding (e.g., for reduced antibody-dependent cellular cytotoxicity(ADCC) induction, increased stability, and/or the ability to formheterodimeric proteins. The variant IgG Fc polypeptides of the presentdisclosure may have broad applicability in companion animaltherapeutics. For example, variant IgG Fc polypeptides may be used inthe design and production of long-acting GLP1 polypeptides for treating,for example, diabetes, obesity, or related indications, in companionanimals, such as canines, felines, and equines. In addition, variant IgGFc polypeptides may be used in the design and production of antibodiesor fusion proteins for treating various disorders in companion animals.

BACKGROUND

IgG Fc plays an important role in Fc-mediated functions thoughinteractions with FcRn, Fc receptor, and C1q. In companion animals,various IgG subtypes possess differences in these functions, which areoften considered when choosing a particular IgG antibody or IgG Fcfusion protein for therapeutic or diagnostic applications. For example,the ability of an IgG subtype to have weak or no measurable bindingaffinity to C1q or CD16 may be advantageous. In addition, IgG Fc'sability to bind Protein A may be useful for purification using a ProteinA affinity purification platform.

However, most IgG Fc subtypes of canine, feline, and equine do notpossess Protein A binding properties, weak or no measurable bindingaffinity to CD16, and weak or no measurable binding affinity to C1q. Forexample, of the four canine IgG Fc subtypes (IgG-A, IgG-B, IgG-C, andIgG-D), only canine IgG-B Fc has appreciable affinity to Protein A.Meanwhile only canine IgG-A Fc and IgG-D Fc have no or weak C1q bindingor CD16 binding. Antibodies and Fc fusion proteins comprising variantIgG Fc polypeptides that have reduced binding to C1q and/or CD16, and/orthat able to bind Protein A are desirable.

Glucagon-like peptide-1 (GLP1) is a potent antihyperglycemic hormone,which plays an important role in regulating blood glucose level. NativeGLP1 has an in vivo half-life of approximately 2 minutes. Long-actingGLP1 polypeptides can be used to treat diabetes and obesity, preventdiabetes, control hyperglycemic conditions, lower lipids, treatconditions that would benefit from lowered blood glucose levels,suppress gastric or intestinal movement, slow gastric emptying, and/ordecrease food intake. There remains a need for long-acting GLP1polypeptides for treating high blood glucose or uncontrollable bloodglucose-induced conditions in companion animals, such as canines,felines, and equines.

SUMMARY OF THE INVENTION Embodiment 1

A polypeptide comprising a variant IgG Fc polypeptide comprising atleast one amino acid modification relative to a wild-type IgG Fcpolypeptide of a companion animal species, wherein the variant IgG Fcpolypeptide has increased binding affinity to Protein A relative to thewild-type IgG Fc polypeptide.

Embodiment 2

A polypeptide comprising a variant IgG Fc polypeptide comprising atleast one amino acid modification relative to a wild-type IgG Fcpolypeptide of a companion animal species, wherein the variant IgG Fcpolypeptide has reduced binding affinity to C1q and/or CD16 relative tothe wild-type IgG Fc polypeptide.

Embodiment 3

The polypeptide of embodiment 1 or embodiment 2, wherein the variant IgGFc polypeptide binds to C1q and/or CD16 with a dissociation constant(K_(d)) of greater than 5×10⁻⁶ M, greater than 1×10⁻⁵ M, greater than5×10⁻⁵ M, greater than 1×10⁻⁴ M, greater than 5×10⁻⁴ M, or greater than1×10⁻³ M, as measured by biolayer interferometry.

Embodiment 4

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide has increased binding affinity to Protein Arelative to the wild-type IgG Fc polypeptide.

Embodiment 5

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide binds to Protein A with a dissociationconstant (K_(d)) of less than 5×10⁻⁶ M, less than 1×10⁻⁶ M, less than5×10⁻⁷ M, less than 1×10⁻⁷ M, less than 5×10⁻⁸ M, less than 1×10⁻⁸ M,less than 5×10⁻⁹ M, less than 1×10⁻⁹ M, less than 5×10⁻¹⁰ M, less than1×10⁻¹⁰ M, less than 5×10⁻¹¹M, less than 1×10⁻¹¹ M, less than 5×10⁻¹² M,or less than 1×10⁻¹² M, as measured by biolayer interferometry.

Embodiment 6

The polypeptide of any one of the preceding embodiments, wherein thecompanion animal species is canine, feline, or equine.

Embodiment 7

The polypeptide of any one of the preceding embodiments, wherein thewild-type IgG Fc polypeptide is

a) a canine IgG-A Fc, IgG-B Fc, IgG-C Fc, or IgG-D Fc;

b) an equine IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5 Fc, IgG6 Fc, orIgG7 Fc; or

c) a feline IgG1a Fc, IgG1b Fc, or IgG2 Fc.

Embodiment 8

A polypeptide comprising a variant IgG Fc polypeptide comprising atleast one amino acid modification to a hinge region relative to awild-type feline or equine IgG Fc polypeptide, wherein the variant IgGFc polypeptide has increased recombinant production and/or increasedhinge disulfide formation relative to the wild-type IgG Fc polypeptide,as determined by SDS-PAGE analysis under reducing and/or nonreducingconditions.

Embodiment 9

The polypeptide of any one the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) at least one amino acid substitution relative to a wild-type felineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises anamino acid substitution at a position corresponding to position 16 ofSEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ IDNO: 118;

b) at least one amino acid substitution relative to a wild-type equineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises anamino acid substitution at a position corresponding to position 3 of SEQID NO: 129; and/or

c) at least one amino acid substitution relative to a wild-type equineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises anamino acid substitution at a position corresponding to position 20 ofSEQ ID NO: 129.

Embodiment 10

The polypeptide of any one the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) at least one amino acid substitution relative to a wild-type felineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises anamino acid substitution at position 16 of SEQ ID NO: 16, SEQ ID NO: 80,SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118;

b) at least one amino acid substitution relative to a wild-type equineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises anamino acid substitution at position 3 of SEQ ID NO: 129; and/or

c) at least one amino acid substitution relative to a wild-type equineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises anamino acid substitution at position 20 of SEQ ID NO: 129.

Embodiment 11

The polypeptide of any one the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) at least one amino acid substitution relative to a wild-type felineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises aproline at a position corresponding to position 16 or at position 16 ofSEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ IDNO: 118;

b) at least one amino acid substitution relative to a wild-type equineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises aserine at a position corresponding to position 3 or at position 3 of SEQID NO: 129; and/or

c) at least one amino acid substitution relative to a wild-type equineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises aproline at a position corresponding to position 20 or at position 20 ofSEQ ID NO: 129.

Embodiment 12

The polypeptide of any one the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises a hinge region or a portion of ahinge region from an IgG Fc polypeptide of a different isotype.

Embodiment 13

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises a hinge region or a portion of ahinge region from a wild-type feline IgG-1a Fc polypeptide, from awild-type feline IgG-1b Fc polypeptide, or from a wild-type equine IgG1Fc polypeptide.

Embodiment 14

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises SEQ ID NO: 19, SEQ ID NO: 125 orSEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: SEQ ID NO:129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQID N: 134, SEQ ID NO: 135.

Embodiment 15

A polypeptide comprising an amino acid sequence of SEQ ID NO: 19, SEQ IDNO: 125 or SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ IDNO: 133, SEQ ID N: 134, SEQ ID NO: 135.

Embodiment 16

A polypeptide comprising a variant IgG2 Fc polypeptide comprising atleast one amino acid substitution relative to a wild-type feline IgG2 Fcpolypeptide, wherein the at least one amino acid substitution is acysteine, and wherein the variant IgG2 Fc polypeptide is capable offorming at least one additional inter-chain disulfide linkage relativeto the wild-type feline IgG2 Fc polypeptide.

Embodiment 17

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises at least one amino acidsubstitution relative to a wild-type feline IgG Fc polypeptide, whereinthe at least one amino acid substitution is a cysteine, and wherein thevariant IgG Fc polypeptide is capable of forming at least one additionalinter-chain disulfide linkage relative to the wild-type feline IgG Fcpolypeptide.

Embodiment 18

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises a cysteine at a positioncorresponding to position 8, position 9, position 10, position 11,position 12, position 13, position 14, position 15, or position 16 ofSEQ ID NO: 16.

Embodiment 19

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises a cysteine at a positioncorresponding to position 14 of SEQ ID NO: 16.

Embodiment 20

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises a cysteine at position 14 of SEQ IDNO: 16.

Embodiment 21

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide is at least 90% identical, at least 95%identical, at least 97% identical, or at least 99% identical to theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ IDNO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 107, SEQ IDNO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112,SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ IDNO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121,SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ IDNO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130,SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ IDNO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 140,SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ IDNO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149,SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ IDNO: 154, SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 157.

Embodiment 22

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises SEQ ID NO: 17.

Embodiment 23

A polypeptide comprising an amino acid sequence of SEQ ID NO: 17.

Embodiment 24

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) an amino acid substitution at a position corresponding to position 21of SEQ ID NO: 1, an amino acid substitution at a position correspondingto position 23 of SEQ ID NO: 1, an amino acid substitution at a positioncorresponding to position 25 of SEQ ID NO: 1, an amino acid substitutionat a position corresponding to position 80 of SEQ ID NO: 1, an aminoacid substitution at a position corresponding to position 205 of SEQ IDNO: 1, and/or an amino acid substitution at a position corresponding toposition 207 of SEQ ID NO: 1;

b) an amino acid substitution at a position corresponding to position 21of SEQ ID NO: 3, an amino acid substitution at a position correspondingto position 23 of SEQ ID NO: 3, and/or an amino acid substitution at aposition corresponding to position 24 of SEQ ID NO: 3;

c) an amino acid substitution at a position corresponding to position 21of SEQ ID NO: 4, an amino acid substitution at a position correspondingto position 23 of SEQ ID NO: 4, an amino acid substitution at a positioncorresponding to position 25 of SEQ ID NO: 4, an amino acid substitutionat a position corresponding to position 80 of SEQ ID NO: 4, and/or anamino acid substitution at a position corresponding to position 207 ofSEQ ID NO: 4;

d) an amino acid substitution at a position corresponding to position 15of SEQ ID NO: 64, and/or an amino acid substitution at a positioncorresponding to position 203 of SEQ ID NO: 64;

e) an amino acid substitution at a position corresponding to position199 of SEQ ID NO: 67, and/or an amino acid substitution at a positioncorresponding to position 200 of SEQ ID NO: 67; and/or

f) an amino acid substitution at a position corresponding to position199 of SEQ ID NO: 68, an amino acid substitution at a positioncorresponding to position 200 of SEQ ID NO: 68, an amino acidsubstitution at a position corresponding to position 201 of SEQ ID NO:68, and/or an amino acid substitution at a position corresponding toposition 202 of SEQ ID NO: 68.

Embodiment 25

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) an amino acid substitution at position 21 of SEQ ID NO: 1, an aminoacid substitution at position 23 of SEQ ID NO: 1, an amino acidsubstitution at position 25 of SEQ ID NO: 1, an amino acid substitutionat position 80 of SEQ ID NO: 1, an amino acid substitution at position205 of SEQ ID NO: 1, and/or an amino acid substitution at position 207of SEQ ID NO: 1;

b) an amino acid substitution at position 21 of SEQ ID NO: 3, an aminoacid substitution at position 23 of SEQ ID NO: 3, and/or an amino acidsubstitution at position 24 of SEQ ID NO: 3;

c) an amino acid substitution at position 21 of SEQ ID NO: 4, an aminoacid substitution at position 23 of SEQ ID NO: 4, an amino acidsubstitution at position 25 of SEQ ID NO: 4, an amino acid substitutionat position 80 of SEQ ID NO: 4, and/or an amino acid substitution atposition 207 of SEQ ID NO: 4;

d) an amino acid substitution at position 15 of SEQ ID NO: 64, and/or anamino acid substitution at position 203 of SEQ ID NO: 64;

e) an amino acid substitution at position 199 of SEQ ID NO: 67, and/oran amino acid substitution at position 200 of SEQ ID NO: 67; and/or

f) an amino acid substitution at position 199 of SEQ ID NO: 68, an aminoacid substitution at position 200 of SEQ ID NO: 68, an amino acidsubstitution at position 201 of SEQ ID NO: 68, and/or an amino acidsubstitution at position 202 of SEQ ID NO: 68.

Embodiment 26

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) a threonine at a position corresponding to position 21 of SEQ ID NO:1, a leucine at a position corresponding to position 23 of SEQ ID NO: 1,an alanine at a position corresponding to position 25 of SEQ ID NO: 1, aglycine at a position corresponding to position 80 of SEQ ID NO: 1, analanine at a position corresponding to position 205 of SEQ ID NO: 1,and/or a histidine at a position corresponding to position 207 of SEQ IDNO: 1;

b) a threonine at a position corresponding to position 21 of SEQ ID NO:3, a leucine at a position corresponding to position 23 of SEQ ID NO: 3,and/or an isoleucine at a position corresponding to position 24 of SEQID NO: 3;

c) a threonine at a position corresponding to position 21 of SEQ ID NO:4, a leucine at a position corresponding to position 23 of SEQ ID NO: 4,an alanine at a position corresponding to position 25 of SEQ ID NO: 4, aglycine at a position corresponding to position 80 of SEQ ID NO: 4,and/or a histidine at a position corresponding to position 207 of SEQ IDNO: 4;

d) a threonine or a valine at a position corresponding to position 15 ofSEQ ID NO: 64, and/or a tyrosine or a valine at a position correspondingto position 203 of SEQ ID NO: 64;

e) a leucine at a position corresponding to position 199 of SEQ ID NO:67, and/or a histidine at a position corresponding to position 200 ofSEQ ID NO: 67; and/or

f) a leucine at a position corresponding to position 199 of SEQ ID NO:68, a histidine at a position corresponding to position 200 of SEQ IDNO: 68, an asparagine at a position corresponding to position 201 of SEQID NO: 68, and/or a histidine at a position corresponding to position202 of SEQ ID NO: 68.

Embodiment 27

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) a threonine at position 21 of SEQ ID NO: 1, a leucine at position 23of SEQ ID NO: 1, an alanine at position 25 of SEQ ID NO: 1, a glycine atposition 80 of SEQ ID NO: 1, an alanine at position 205 of SEQ ID NO: 1,and/or a histidine at position 207 of SEQ ID NO: 1;

b) a threonine at position 21 of SEQ ID NO: 3, a leucine at position 23of SEQ ID NO: 3, and/or an isoleucine at position 24 of SEQ ID NO: 3;

c) a threonine at a position 21 of SEQ ID NO: 4, a leucine at position23 of SEQ ID NO: 4, an alanine at position 25 of SEQ ID NO: 4, a glycineat position 80 of SEQ ID NO: 4, and/or a histidine at position 207 ofSEQ ID NO: 4;

d) a threonine or a valine at position 15 of SEQ ID NO: 64, and/or atyrosine or a valine at position 203 of SEQ ID NO: 64;

e) a leucine at position 199 of SEQ ID NO: 67, and/or a histidine atposition 200 of SEQ ID NO: 67; and/or

f) a leucine at position 199 of SEQ ID NO: 68, a histidine at position200 of SEQ ID NO: 68, an asparagine at position 201 of SEQ ID NO: 68,and/or a histidine at position 202 of SEQ ID NO: 68.

Embodiment 28

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises an amino acid sequence of:

a) SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 60, SEQ ID NO:61, SEQ ID NO: 62, or SEQ ID NO: 84; or

b) SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, or SEQ IDNO: 76.

Embodiment 29

A polypeptide comprising an amino sequence of SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:84, SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, or SEQID NO: 76.

Embodiment 30

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) an amino acid substitution at a position corresponding to position 93of SEQ ID NO: 2, or an amino acid substitution at a positioncorresponding to position 93 of SEQ ID NO: 3;

b) an amino acid substitution at a position corresponding to position 87of SEQ ID NO: 63, an amino acid substitution at a position correspondingto position 87 of SEQ ID NO: 65, an amino acid substitution at aposition corresponding to position 87 of SEQ ID NO: 66, or an amino acidsubstitution at a position corresponding to position 87 of SEQ ID NO:69; or

c) an amino acid substitution at a position corresponding to position198 of SEQ ID NO: 80, or an amino acid substitution at a positioncorresponding to position 198 of SEQ ID NO: 81.

Embodiment 31

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) an amino acid substitution at position 93 of SEQ ID NO: 2, or anamino acid substitution at position 93 of SEQ ID NO: 3;

b) an amino acid substitution at position 87 of SEQ ID NO: 63, an aminoacid substitution at position 87 of SEQ ID NO: 65, an amino acidsubstitution at position 87 of SEQ ID NO: 66, or an amino acidsubstitution at position 87 of SEQ ID NO: 69; or

c) an amino acid substitution at position 198 of SEQ ID NO: 80, or anamino acid substitution at position 198 of SEQ ID NO: 81.

Embodiment 32

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) an arginine at a position corresponding to position 93 of SEQ ID NO:2, or an arginine at a position corresponding to position 93 of SEQ IDNO: 3;

b) a serine at a position corresponding to position 87 of SEQ ID NO: 63,a serine substitution at a position corresponding to position 87 of SEQID NO: 65, a serine at a position corresponding to position 87 of SEQ IDNO: 66, or a serine at a position corresponding to position 87 of SEQ IDNO: 69; or

c) an alanine at a position corresponding to position 198 of SEQ ID NO:80, or an alanine at a position corresponding to position 198 of SEQ IDNO: 81.

Embodiment 33

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) an arginine at position 93 of SEQ ID NO: 2, or an arginine atposition 93 of SEQ ID NO: 3;

b) a serine at position 87 of SEQ ID NO: 63, a serine at position 87 ofSEQ ID NO: 65, a serine at position 87 of SEQ ID NO: 66, or a serine atposition 87 of SEQ ID NO: 69; or

c) an alanine at position 198 of SEQ ID NO: 80, or alanine at position198 of SEQ ID NO: 81.

Embodiment 34

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises the amino acid sequence of:

a) SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 84; or

b) SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 77; or

c) SEQ ID NO: 82 or SEQ ID NO: 83.

Embodiment 35

A polypeptide comprising an amino sequence of SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO: 84, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ IDNO: 77, SEQ ID NO: 82, or SEQ ID NO: 83.

Embodiment 36

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) an amino acid substitution at a position corresponding to position 5of SEQ ID NO: 2, an amino acid substitution at a position correspondingto position 38 of SEQ ID NO: 2, an amino acid substitution at a positioncorresponding to position 39 of SEQ ID NO: 2, an amino acid substitutionat a position corresponding to position 97 of SEQ ID NO: 2, and/or anamino acid substitution at a position corresponding to position 98 ofSEQ ID NO: 2; or

b) an amino acid substitution at a position corresponding to position 5of SEQ ID NO: 3, an amino acid substitution at a position correspondingto position 38 of SEQ ID NO: 3, an amino acid substitution at a positioncorresponding to position 39 of SEQ ID NO: 3, an amino acid substitutionat a position corresponding to position 97 of SEQ ID NO: 3, and/or anamino acid substitution at a position corresponding to position 98 ofSEQ ID NO: 3.

Embodiment 37

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) an amino acid substitution at position 5 of SEQ ID NO: 2, an aminoacid substitution at position 38 of SEQ ID NO: 2, an amino acidsubstitution at position 39 of SEQ ID NO: 2, an amino acid substitutionat position 97 of SEQ ID NO: 2, and/or an amino acid substitution atposition 98 of SEQ ID NO: 2; or

b) an amino acid substitution at position 5 of SEQ ID NO: 3, an aminoacid substitution at position 38 of SEQ ID NO: 3, an amino acidsubstitution at position 39 of SEQ ID NO: 3, an amino acid substitutionat position 97 of SEQ ID NO: 3, and/or an amino acid substitution atposition 98 of SEQ ID NO: 3.

Embodiment 38

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) a proline at a position corresponding to position 5 of SEQ ID NO: 2,a glycine at a position corresponding to position 38 of SEQ ID NO: 2, anarginine at a position corresponding to position 39 of SEQ ID NO: 2, anisoleucine at a position corresponding to position 97 of SEQ ID NO: 2,and/or a glycine at a position corresponding to position 98 of SEQ IDNO: 2; or

b) a proline at a position corresponding to position 5 of SEQ ID NO: 3,a glycine at a position corresponding to position 38 of SEQ ID NO: 3, anarginine at a position corresponding to position 39 of SEQ ID NO: 3, anisoleucine at a position corresponding to position 97 of SEQ ID NO: 3,and/or a glycine at a position corresponding to position 98 of SEQ IDNO: 3.

Embodiment 39

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) a proline at position 5 of SEQ ID NO: 2, a glycine at position 38 ofSEQ ID NO: 2, an arginine at position 39 of SEQ ID NO: 2, an isoleucineat position 97 of SEQ ID NO: 2, and/or a glycine at position 98 of SEQID NO: 2; or

b) a proline at position 5 of SEQ ID NO: 3, a glycine at position 38 ofSEQ ID NO: 3, an arginine at position 39 of SEQ ID NO: 3, an isoleucineat position 97 of SEQ ID NO: 3, and/or a glycine at position 98 of SEQID NO: 3.

Embodiment 40

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises an amino acid sequence of:

a) SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, or SEQ IDNO: 147; or

b) SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQID NO: 152, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, or SEQ IDNO: 157.

Embodiment 41

A polypeptide comprising an amino sequence of SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 154, SEQID NO: 155, SEQ ID NO: 156, or SEQ ID NO: 157.

Embodiment 42

A polypeptide comprising a variant IgG Fc polypeptide comprising:

a) a tyrosine or a tryptophan at a position corresponding to position138 of SEQ ID NO: 1, a tyrosine or a tryptophan at a positioncorresponding to position 137 of SEQ ID NO: 2, a tyrosine or atryptophan at a position corresponding to position 137 of SEQ ID NO: 3,or a tyrosine or a tryptophan at a position corresponding to position138 of SEQ ID NO: 4; or

b) a tyrosine or a tryptophan at a position corresponding to position154 of SEQ ID NO: 16, a tyrosine or a tryptophan at a positioncorresponding to position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or atyrosine or a tryptophan at a position corresponding to position 154 ofSEQ ID NO: 81 or SEQ ID NO: 118.

Embodiment 43

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) a tyrosine or a tryptophan at position 138 of SEQ ID NO: 1, atyrosine or a tryptophan at position 137 of SEQ ID NO: 2, a tyrosine ora tryptophan at position 137 of SEQ ID NO: 3, or a tyrosine or atryptophan at position 138 of SEQ ID NO: 4; or

b) a tyrosine or a tryptophan at position 154 of SEQ ID NO: 16, atyrosine or a tryptophan at position 154 of SEQ ID NO: 80 or SEQ ID NO:117, or a tyrosine or a tryptophan at a position corresponding toposition 154 of SEQ ID NO: 81 or SEQ ID NO: 118.

Embodiment 44

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises an amino acid sequence of SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO:121, or SEQ ID NO: 123.

Embodiment 45

A polypeptide comprising an amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111,SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 121, or SEQID NO: 123.

Embodiment 46

A contiguous polypeptide comprising the polypeptide of any one of thepreceding embodiments and a glucagon-like peptide-1 (GLP1) polypeptide.

Embodiment 47

A contiguous polypeptide comprising the polypeptide of any one of thepreceding embodiments and a glucagon polypeptide.

Embodiment 48

A polypeptide comprising a variant IgG Fc polypeptide comprising:

a) a serine at a position corresponding to position 138 of SEQ ID NO: 1,a serine at a position corresponding to position 137 of SEQ ID NO: 2, aserine at a position corresponding to position 137 of SEQ ID NO: 3, aserine at a position corresponding to position 138 of SEQ ID NO: 4, aserine at a position corresponding to position 154 of SEQ ID NO: 16, aserine at a position corresponding to position 154 of SEQ ID NO: 80 orSEQ ID NO: 117, or a serine at a position corresponding to position 154of SEQ ID NO: 81 or SEQ ID NO: 118;

b) an alanine at a position corresponding to position 140 of SEQ ID NO:1, an alanine at a position corresponding to position 139 of SEQ ID NO:2, an alanine at a position corresponding to position 139 of SEQ ID NO:3, an alanine at a position corresponding to position 140 of SEQ ID NO:4, an alanine at a position corresponding to position 156 of SEQ ID NO:16, an alanine at a position corresponding to position 156 of SEQ ID NO:80 or SEQ ID NO: 117, or an alanine at a position corresponding toposition 156 of SEQ ID NO: 81 or SEQ ID NO: 118; and/or

c) a threonine at a position corresponding to position 181 of SEQ ID NO:1, a threonine at a position corresponding to position 180 of SEQ ID NO:2, a threonine at a position corresponding to position 180 of SEQ ID NO:3, a threonine at a position corresponding to position 181 of SEQ ID NO:4, a threonine at a position corresponding to position 197 of SEQ ID NO:16, a threonine at a position corresponding to position 197 of SEQ IDNO: 80 or SEQ ID NO: 117, or a threonine at a position corresponding toposition 197 of SEQ ID NO: 81 or SEQ ID NO: 118.

Embodiment 49

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises:

a) a serine at position 138 of SEQ ID NO: 1, a serine at position 137 ofSEQ ID NO: 2, a serine at position 137 of SEQ ID NO: 3, a serine atposition 138 of SEQ ID NO: 4, a serine at position 154 of SEQ ID NO: 16,a serine at position 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a serineat position 154 of SEQ ID NO: 81 or SEQ ID NO: 118;

b) an alanine at position 140 of SEQ ID NO: 1, an alanine at position139 of SEQ ID NO: 2, an alanine at position 139 of SEQ ID NO: 3, analanine at position 140 of SEQ ID NO: 4, an alanine at position 156 ofSEQ ID NO: 16, an alanine at position 156 of SEQ ID NO: 80 or SEQ ID NO:117, or an alanine at position 156 of SEQ ID NO: 81 or SEQ ID NO: 118;and/or;

c) a threonine at position 181 of SEQ ID NO: 1, a threonine at position181 of SEQ ID NO: 2, a threonine at position 181 of SEQ ID NO: 3, athreonine at position 181 of SEQ ID NO: 4, a threonine at position 197of SEQ ID NO: 16, a threonine at position 197 of SEQ ID NO: 80 or SEQ IDNO: 117, or a threonine at position 197 of SEQ ID NO: 81 or SEQ ID NO:118.

Embodiment 50

The polypeptide of any one of the preceding embodiments, wherein thevariant IgG Fc polypeptide comprises an amino acid sequence of SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO:122, or SEQ ID NO: 124.

Embodiment 51

A polypeptide comprising an amino acid sequence of SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112,SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 122, or SEQID NO: 124.

Embodiment 52

The polypeptide of any one of the preceding embodiments, wherein thepolypeptide is glycoslylated.

Embodiment 53

The polypeptide of any one of embodiments 1 to 51, wherein thepolypeptide is a glycosylated.

Embodiment 54

A contiguous polypeptide comprising the polypeptide of any one ofembodiments 48 to 53 and a glucagon-like peptide-1 (GLP1) polypeptide.

Embodiment 55

A contiguous polypeptide comprising the polypeptide of any one ofembodiments 48 to 53 and a glucagon polypeptide.

Embodiment 56

A heterodimeric protein comprising the contiguous polypeptide ofembodiment 46 and the contiguous polypeptide of embodiment 54.

Embodiment 57

A heterodimeric protein comprising the contiguous polypeptide ofembodiment 47 and the contiguous polypeptide of embodiment 55.

Embodiment 58

The contiguous polypeptide or heterodimeric protein of any one ofembodiments 46, 47, or 54 to 57, wherein the GLP1 polypeptide is awild-type GLP1 polypeptide, optionally comprising the amino acidsequence of SEQ ID NO: 85.

Embodiment 59

The contiguous polypeptide or heterodimeric protein of any one ofembodiments 46, 47, or 54 to 58, wherein the GLP1 polypeptide is avariant GLP1 polypeptide.

Embodiment 60

The contiguous polypeptide or heterodimeric protein of any one ofembodiments 46, 47, or 54 to 59, wherein the GLP1 polypeptide comprisesthe amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98,or SEQ ID NO: 99.

Embodiment 61

The contiguous polypeptide or heterodimeric protein of any one ofembodiments 46, 47, or 54 to 60, wherein the glucagon polypeptide is awild-type glucagon polypeptide, optionally comprising the amino acidsequence of SEQ ID NO: 21.

Embodiment 62

The contiguous polypeptide or heterodimeric protein of any one ofembodiments 46, 47, or 54 to 61, wherein the glucagon polypeptide is avariant glucagon polypeptide.

Embodiment 63

A heterodimeric protein comprising:

i) a first variant canine IgG Fc polypeptide comprising at least oneamino acid modification relative to a first wild-type canine IgG Fcpolypeptide and a second variant canine IgG Fc polypeptide comprising atleast one amino acid modification relative to a second wild-type canineIgG Fc polypeptide; orii) a first variant feline IgG Fc polypeptide comprising at least oneamino acid modification relative to a first wild-type feline IgG Fcpolypeptide and a second variant feline IgG Fc polypeptide comprising atleast one amino acid modification relative to a second wild-type felineIgG Fc polypeptide, wherein:

a) the first variant canine IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 138 of SEQ ID NO:1, position 137 of SEQ ID NO: 2, position 137 of SEQ ID NO: 3, orposition 138 of SEQ ID NO: 4;

b) the second variant canine IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 138, position 140,and/or position 181 of SEQ ID NO: 1, position 137, position 139, and/orposition 180 of SEQ ID NO: 2, position 137, position 139, and/orposition 180 of SEQ ID NO: 3, or position 138, position 140, and/orposition 181 of SEQ ID NO: 4;

c) the first variant feline IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 154 of SEQ ID NO:6, of SEQ ID NO: 80, of SEQ ID NO: 81, of SEQ ID NO: 117, or of SEQ IDNO: 118; and/or

d) the second variant feline IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 154, position 156,and/or position 197 of SEQ ID NO: 6, of SEQ ID NO: 80, of SEQ ID NO: 81,of SEQ ID NO: 117, or of SEQ ID NO: 118.

Embodiment 64

The heterodimeric protein of embodiment 63, wherein the first wild-typecanine IgG Fc polypeptide and the second wild-type canine IgG Fcpolypeptide are from the same IgG subtype and/or the first wild-typefeline IgG Fc polypeptide and the second wild-type feline IgG Fcpolypeptide are from the same IgG subtype.

Embodiment 65

The heterodimeric protein of embodiment 63, wherein the first wild-typecanine IgG Fc polypeptide and the second wild-type canine IgG Fcpolypeptide are from a different IgG subtype and/or the first wild-typefeline IgG Fc polypeptide and the second wild-type feline IgG Fcpolypeptide are from the same IgG subtype.

Embodiment 66

The heterodimeric protein of any one of embodiments 63 to 65, wherein:

a) the first variant canine IgG Fc polypeptide comprises a tyrosine ortryptophan at a position corresponding to position 138 of SEQ ID NO: 1,position 137 of SEQ ID NO: 2, position 137 of SEQ ID NO: 3, or position138 of SEQ ID NO: 4; and/or

b) the first variant feline IgG Fc polypeptide comprises a tryptophan ata position corresponding to position 154 of SEQ ID NO: 6, of SEQ ID NO:80, of SEQ ID NO: 81, of SEQ ID NO: 117, or of SEQ ID NO: 118.

Embodiment 67

The heterodimeric protein of any one of embodiments 63 to 66, wherein:

a) the second variant canine IgG Fc polypeptide comprises a serine at aposition corresponding to position 138, an alanine at a positioncorresponding to position 140, and/or a threonine at a positioncorresponding to position 181 of SEQ ID NO: 1, a serine at a positioncorresponding to position 137, an alanine at a position corresponding toposition 139, and/or a threonine at a position corresponding to position180 of SEQ ID NO: 2, a serine at a position corresponding to position137, an alanine at a position corresponding to position 139, and/or athreonine at a position corresponding to position 180 of SEQ ID NO: 3,and/or a serine at a position corresponding to position 138, an alanineat a position corresponding to position 140, and/or a threonine at aposition corresponding to position 181 of SEQ ID NO: 4; and/or

b) the second variant feline IgG Fc polypeptide comprises a serine at aposition corresponding to position 154, an alanine at a positioncorresponding to position 156, and/or a threonine at a positioncorresponding to position 197 of SEQ ID NO: 6, of SEQ ID NO: 80, of SEQID NO: 81, of SEQ ID NO: 117, or of SEQ ID NO: 118.

Embodiment 68

The heterodimeric protein of any one of embodiments 63 to 67, wherein:

a) the first variant canine IgG Fc polypeptide comprises an amino acidsequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, or SEQ ID NO: 115;and/or

b) the first variant feline IgG Fc polypeptide comprises an amino acidsequence of SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123.

Embodiment 69

The heterodimeric protein of any one of embodiments 63 to 68, wherein:

a) the second variant canine IgG Fc polypeptide comprises an amino acidsequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, or SEQ ID NO: 116;and/or

b) the second variant feline IgG Fc polypeptide comprises an amino acidsequence of SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO: 123.

Embodiment 70

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 69, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises at least one additional amino acidmodification relative to a wild-type IgG Fc polypeptide and hasincreased binding affinity to Protein A relative to the wild-type IgG Fcpolypeptide.

Embodiment 71

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 70, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises:

a) an amino acid substitution at a position corresponding to position 21of SEQ ID NO: 1, an amino acid substitution at a position correspondingto position 23 of SEQ ID NO: 1, an amino acid substitution at a positioncorresponding to position 25 of SEQ ID NO: 1, an amino acid substitutionat a position corresponding to position 80 of SEQ ID NO: 1, an aminoacid substitution at a position corresponding to position 205 of SEQ IDNO: 1, and/or an amino acid substitution at a position corresponding toposition 207 of SEQ ID NO: 1;

b) an amino acid substitution at a position corresponding to position 21of SEQ ID NO: 3, an amino acid substitution at a position correspondingto position 23 of SEQ ID NO: 3, and/or an amino acid substitution at aposition corresponding to position 24 of SEQ ID NO: 3; or

c) an amino acid substitution at a position corresponding to position 21of SEQ ID NO: 4, an amino acid substitution at a position correspondingto position 23 of SEQ ID NO: 4, an amino acid substitution at a positioncorresponding to position 25 of SEQ ID NO: 4, an amino acid substitutionat a position corresponding to position 80 of SEQ ID NO: 4, and/or anamino acid substitution at a position corresponding to position 207 ofSEQ ID NO: 4.

Embodiment 72

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 71, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises:

a) an amino acid substitution at position 21 of SEQ ID NO: 1, an aminoacid substitution at position 23 of SEQ ID NO: 1, an amino acidsubstitution at position 25 of SEQ ID NO: 1, an amino acid substitutionat position 80 of SEQ ID NO: 1, an amino acid substitution at position205 of SEQ ID NO: 1, and/or an amino acid substitution at position 207of SEQ ID NO: 1;

b) an amino acid substitution at position 21 of SEQ ID NO: 3, an aminoacid substitution at position 23 of SEQ ID NO: 3, and/or an amino acidsubstitution at position 24 of SEQ ID NO: 3; or

c) an amino acid substitution at position 21 of SEQ ID NO: 4, an aminoacid substitution at position 23 of SEQ ID NO: 4, an amino acidsubstitution at position 25 of SEQ ID NO: 4, an amino acid substitutionat position 80 of SEQ ID NO: 4, and/or an amino acid substitution atposition 207 of SEQ ID NO: 4.

Embodiment 73

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 72, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises:

a) a threonine at a position corresponding to position 21 of SEQ ID NO:1, a leucine at a position corresponding to position 23 of SEQ ID NO: 1,an alanine at a position corresponding to position 25 of SEQ ID NO: 1, aglycine at a position corresponding to position 80 of SEQ ID NO: 1, analanine at a position corresponding to position 205 of SEQ ID NO: 1,and/or a histidine at a position corresponding to position 207 of SEQ IDNO: 1;

b) a threonine at a position corresponding to position 21 of SEQ ID NO:3, a leucine at a position corresponding to position 23 of SEQ ID NO: 3,and/or an isoleucine at a position corresponding to position 24 of SEQID NO: 3; or

c) a threonine at a position corresponding to position 21 of SEQ ID NO:4, a leucine at a position corresponding to position 23 of SEQ ID NO: 4,an alanine at a position corresponding to position 25 of SEQ ID NO: 4, aglycine at a position corresponding to position 80 of SEQ ID NO: 4,and/or a histidine at a position corresponding to position 207 of SEQ IDNO: 4.

Embodiment 74

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 73, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises:

a) a threonine at position 21 of SEQ ID NO: 1, a leucine at position 23of SEQ ID NO: 1, an alanine at position 25 of SEQ ID NO: 1, a glycine atposition 80 of SEQ ID NO: 1, an alanine at position 205 of SEQ ID NO: 1,and/or a histidine at position 207 of SEQ ID NO: 1;

b) a threonine at position 21 of SEQ ID NO: 3, a leucine at position 23of SEQ ID NO: 3, and/or an isoleucine at position 24 of SEQ ID NO: 3; or

c) a threonine at position 21 of SEQ ID NO: 4, a leucine at position 23of SEQ ID NO: 4, an alanine at position 25 of SEQ ID NO: 4, a glycine atposition 80 of SEQ ID NO: 4, and/or a histidine at position 207 of SEQID NO: 4.

Embodiment 75

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 74, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises at least one additional amino acidmodification relative to a wild-type IgG Fc polypeptide and hasdecreased binding affinity to CD16 relative to the wild-type IgG Fcpolypeptide.

Embodiment 76

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 75, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises:

a) an amino acid substitution at a position corresponding to position 5of SEQ ID NO: 2, an amino acid substitution at a position correspondingto position 38 of SEQ ID NO: 2, an amino acid substitution at a positioncorresponding to position 39 of SEQ ID NO: 2, an amino acid substitutionat a position corresponding to position 97 of SEQ ID NO: 2, and/or anamino acid substitution at a position corresponding to position 98 ofSEQ ID NO: 2; or

b) an amino acid substitution at a position corresponding to position 5of SEQ ID NO: 3, an amino acid substitution at a position correspondingto position 38 of SEQ ID NO: 3, an amino acid substitution at a positioncorresponding to position 39 of SEQ ID NO: 3, an amino acid substitutionat a position corresponding to position 97 of SEQ ID NO: 3, and/or anamino acid substitution at a position corresponding to position 98 ofSEQ ID NO: 3.

Embodiment 77

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 76, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises:

a) an amino acid substitution at position 5 of SEQ ID NO: 2, an aminoacid substitution at position 38 of SEQ ID NO: 2, an amino acidsubstitution at position 39 of SEQ ID NO: 2, an amino acid substitutionat position 97 of SEQ ID NO: 2, and/or an amino acid substitution atposition 98 of SEQ ID NO: 2; or

b) an amino acid substitution at position 5 of SEQ ID NO: 3, an aminoacid substitution at position 38 of SEQ ID NO: 3, an amino acidsubstitution at position 39 of SEQ ID NO: 3, an amino acid substitutionat position 97 of SEQ ID NO: 3, and/or an amino acid substitution atposition 98 of SEQ ID NO: 3.

Embodiment 78

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 77, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises:

a) a proline at a position corresponding to position 5 of SEQ ID NO: 2,a glycine at a position corresponding to position 38 of SEQ ID NO: 2, anarginine at a position corresponding to position 39 of SEQ ID NO: 2, anisoleucine at a position corresponding to position 97 of SEQ ID NO: 2,and/or a glycine at a position corresponding to position 98 of SEQ IDNO: 2; or

b) a proline at a position corresponding to position 5 of SEQ ID NO: 3,a glycine at a position corresponding to position 38 of SEQ ID NO: 3, anarginine at a position corresponding to position 39 of SEQ ID NO: 3, anisoleucine at a position corresponding to position 97 of SEQ ID NO: 3,and/or a glycine at a position corresponding to position 98 of SEQ IDNO: 3.

Embodiment 79

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 78, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises:

a) a proline at position 5 of SEQ ID NO: 2, a glycine at position 38 ofSEQ ID NO: 2, an arginine at position 39 of SEQ ID NO: 2, an isoleucineat position 97 of SEQ ID NO: 2, and/or a glycine at position 98 of SEQID NO: 2; or

b) a proline at position 5 of SEQ ID NO: 3, a glycine at position 38 ofSEQ ID NO: 3, an arginine at position 39 of SEQ ID NO: 3, an isoleucineat position 97 of SEQ ID NO: 3, and/or a glycine at position 98 of SEQID NO: 3.

Embodiment 80

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 79, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises at least one additional amino acidmodification relative to a wild-type canine IgG Fc polypeptide and hasdecreased binding affinity to C1q relative to the wild-type canine IgGFc polypeptide.

Embodiment 81

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 80, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises an amino acid substitution at aposition corresponding to position 93 of SEQ ID NO: 2, or an amino acidsubstitution at a position corresponding to position 93 of SEQ ID NO: 3.

Embodiment 82

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 81, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises an amino acid substitution atposition 93 of SEQ ID NO: 2, or an amino acid substitution at position93 of SEQ ID NO: 3.

Embodiment 83

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 82, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises an arginine at a positioncorresponding to position 93 of SEQ ID NO: 2, or an arginine at aposition corresponding to position 93 of SEQ ID NO: 3.

Embodiment 84

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 42 to 83, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises an arginine at position 93 of SEQID NO: 2, or an arginine at position 93 of SEQ ID NO: 3.

Embodiment 85

The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of embodiments 1 to 84, wherein the polypeptide is anantibody, an antibody fusion, or a fusion polypeptide.

Embodiment 86

A contiguous polypeptide comprising:

a) a first glucagon-like peptide-1 (GLP1) polypeptide (GLP1A);

b) a first linker (L1);

c) an Fc polypeptide (Fc) of a companion animal species;

d) optionally, a second linker (L2); and

e) optionally, a second GLP1 polypeptide (GLP1B).

Embodiment 87

The contiguous polypeptide of embodiment 65 comprising:

GLP1A-L1-Fc; or  formula (I):

Fc-L1GLP1A.  formula (II):

Embodiment 88

The contiguous polypeptide of embodiment 65 comprising:

GLP1A-L1-Fc-L2-GLP1B.  formula (III):

Embodiment 89

The contiguous polypeptide of any one of embodiments 86 to 88, whereinGLP1B, if present, comprises the same amino acid sequence as GLP1A.

Embodiment 90

A contiguous polypeptide comprising:

a) a glucagon-like peptide-1 (GLP1) polypeptide;

b) a first linker (L1);

c) an Fc polypeptide (Fc);

d) a second linker (L2); and

e) a glucagon polypeptide (Gluc).

Embodiment 91

The contiguous polypeptide of embodiment 90 comprising:

GLP1-L1-Fc-L2-Gluc; or  Formula (IV):

Gluc-L1-Fc-L2-GLP1.  Formula (V):

Embodiment 92

The contiguous polypeptide of any one of embodiments 86 to 91, whereinGLP1A, GLP1, and/or GLP1B, if present, comprises a wild-type GLP1polypeptide.

Embodiment 93

The contiguous polypeptide of any one of embodiments 86 to 92, whereinGLP1A, GLP1, and/or GLP1B, if present, comprises a variant GLP1polypeptide.

Embodiment 94

The contiguous polypeptide of any one of embodiments 86 to 93, whereinGLP1A, GLP1, and/or GLP1B, if present, comprises an amino acid sequenceof SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98, or SEQ IDNO: 99.

Embodiment 95

The contiguous polypeptide of any one of embodiments 86 to 94, whereinthe glucagon polypeptide comprises a wild-type glucagon polypeptide,optionally comprising the amino acid sequence of SEQ ID NO: 21.

Embodiment 96

The contiguous polypeptide of any one of embodiments 86 to 95, whereinthe glucagon polypeptide is a variant glucagon polypeptide.

Embodiment 97

The contiguous polypeptide of any one of embodiments 86 to 96, whereinthe Fc polypeptide is a human IgG Fc.

Embodiment 98

The contiguous polypeptide of any one of embodiments 86 to 97, whereinthe Fc polypeptide is a human IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc.

Embodiment 99

The contiguous polypeptide of any one of embodiments 86 to 98, whereinthe Fc polypeptide is an Fc of a companion animal species.

Embodiment 100

The contiguous polypeptide of any one of embodiments 86 to 97 or 99,wherein the Fc polypeptide comprises:

a) a canine IgG-A Fc, IgG-B Fc, IgG-C Fc, or IgG-D Fc;

b) an equine IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5 Fc, IgG6 Fc, orIgG7 Fc; or

c) a feline IgG1a Fc, IgG1b Fc, or IgG2 Fc.

Embodiment 101

The contiguous polypeptide of any one of embodiments 86 to 100, whereinthe Fc polypeptide is a wild-type IgG Fc polypeptide.

Embodiment 102

The contiguous polypeptide of any one of embodiments 86 to 100, whereinthe Fc polypeptide is a variant IgG Fc polypeptide.

Embodiment 103

The contiguous polypeptide of any one of embodiments 86 to 102, whereinthe Fc polypeptide comprises the polypeptide, the contiguouspolypeptide, or the heterodimeric protein of any one of embodiments 1 to84.

Embodiment 104

The contiguous polypeptide of any one of embodiments 85 to 102, whereinthe contiguous polypeptide has a longer serum half-life than a wild-typeGLP1 polypeptide.

Embodiment 105

The contiguous polypeptide of any one of embodiments 86 to 104, whereinL1 and L2, if present, each independently is a flexible linker.

Embodiment 106

The contiguous polypeptide of any one of embodiments 86 to 105, whereinthe amino acid sequence of L1 and L2, if present, each independentlycomprises 100%, at least 95%, at least 90%, at least 85% serine and/orglycine amino acid residues.

Embodiment 107

The contiguous polypeptide of any one of embodiments 86 to 106, whereinthe contiguous polypeptide comprises an extension at its C-terminus.

Embodiment 108

The contiguous polypeptide of any one of embodiments 86 to 107, whereinthe contiguous polypeptide comprises a glycine residue, two glycineresidues, three glycine residues, four glycine residues, five glycineresidues, six glycine residues, seven glycine residues, eight glycineresidues, or greater than eight glycine residues at its C-terminus.

Embodiment 109

The contiguous polypeptide of any one of embodiments 86 to 108, whereinthe contiguous polypeptide comprises an amino acid sequence of SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95 at its C-terminus.

Embodiment 110

The contiguous polypeptide of any one of embodiments 86 to 109, whereinthe contiguous polypeptide comprises:

a) the amino acid sequence of SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO:25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ IDNO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39;SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO:44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47, SEQ ID NO: 96, SEQ IDNO: 97, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO:106; or

b) the amino acid sequence of SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO:54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; or SEQID NO: 59.

Embodiment 111

A polypeptide comprising an amino acid sequence of SEQ ID NO: 23; SEQ IDNO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33;SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO:38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ IDNO: 43; SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47, SEQID NO: 96, SEQ ID NO: 97, SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54;SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO:59; SEQ ID NO: 103; SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106.

Embodiment 112

The polypeptide, the heterodimeric protein, or the contiguouspolypeptide of any one of the preceding embodiments, wherein the atleast one amino acid modification or substitution comprises an aminoacid substitution with an amino acid derivative.

Embodiment 113

An isolated nucleic acid encoding the polypeptide, the heterodimericprotein, or the contiguous polypeptide of any one of the precedingembodiments.

Embodiment 114

A host cell comprising the nucleic acid of embodiment 113.

Embodiment 115

A method of producing a polypeptide comprising culturing the host cellof embodiment 114 and isolating the polypeptide.

Embodiment 116

A pharmaceutical composition comprising the polypeptide, theheterodimeric protein, or the contiguous polypeptide of any one ofembodiments 1 to 112, and a pharmaceutically acceptable carrier.

Embodiment 117

A method of increasing production of cAMP in a cell, the methodcomprising exposing the cell to the polypeptide, the heterodimericprotein, the contiguous polypeptide, or the pharmaceutical compositionof any one of embodiments 1 to 112 or 116 under conditions permissivefor binding of the polypeptide, heterodimeric protein, or contiguouspolypeptide to GLP1R.

Embodiment 118

The method of embodiment 117, wherein the cell is exposed to thepolypeptide, heterodimeric protein, contiguous polypeptide, or thepharmaceutical composition ex vivo.

Embodiment 119

The method of embodiment 117, wherein the cell is exposed to thepolypeptide, heterodimeric protein, contiguous polypeptide, or thepharmaceutical composition in vivo.

Embodiment 120

The method of any one of embodiments 118 to 119, wherein the cell is ahuman cell, a canine cell, a feline cell, or an equine cell.

Embodiment 121

A method of delivering a polypeptide to a subject comprisingadministering the polypeptide, the heterodimeric protein, the contiguouspolypeptide, or the pharmaceutical composition of any one of embodiments1 to 112 or 116 parenterally.

Embodiment 122

A method of delivering a polypeptide to a subject comprisingadministering the polypeptide, the heterodimeric protein, the contiguouspolypeptide, or the pharmaceutical composition of any one of embodiments1 to 112 or 116 by an intramuscular route, an intraperitoneal route, anintracerobrospinal route, a subcutaneous route, an intra-arterial route,an intrasynovial route, an intrathecal route, or an inhalation route.

Embodiment 123

A method of treating a subject having diabetes or obesity, the methodcomprising administering to the subject a therapeutically effectiveamount of the polypeptide, the heterodimeric protein, the contiguouspolypeptide, or the pharmaceutical composition of any one of embodiments1 to 112 or 116.

Embodiment 124

The method of embodiment 123, comprising administering insulin, a DPP4inhibitor, a SGLT2 inhibitor, a biguanides sulfonylureas meglitinidederivative, an alpha-glucosidase inhibitor, a thiazolidinedion (TZD), anamylinomimetic, a bile acid sequestrant, a dopamine agonist.

Embodiment 125

The method of any one of embodiments 121 to 124, wherein the subject isa human subject.

Embodiment 126

The method of any one of embodiments 121 to 124, wherein the subject isa companion animal species.

Embodiment 127

The method of embodiment 126, wherein the companion animal species iscanine, equine, or feline.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of canine IgG-A, B, C, and D Fc sequences. Theboxes indicate the regions likely in contact with Protein A.

FIG. 2A shows an SDS-PAGE analysis of GLP1-G8/GLP-2G_III_WTfeIgG2 (SEQID NO: 23; “GLP1 A variant” in this figure) and GLP1-G8_I_WTfeIgG2 (SEQID NO: 24; “GLP1 B variant” in this figure) having wild-type feline IgG2hinge with one disulfide bond in the absence and presence of reducingagent (DTT).

FIG. 2B shows an SDS-PAGE analysis of GLP1-G8/GLP-2G_III_VARfeIgG2 (SEQID NO: 25; “GLP1 MA variant” in this figure) of GLP1-G8_I_VARfeIgG2 (SEQID NO: 26; “GLP1 MB variant” in this figure) having variant feline IgG2hinge with two disulfide bonds in the absence and presence of reducingagent (DTT).

FIG. 3 shows a cAMP CHO-K1 GLP1R Bioassay to evaluate activity ofGLP1-G8/GLP1-2G_III_VARfeIgG2 (SEQ ID NO: 25) and GLP1-G8_I_VARfeIgG2(SEQ ID NO: 26) compared to controls (GLP1 (7-37) and Extendin-4).

FIG. 4 shows a cell-based bioassay to evaluate activity ofGLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) (“GLP1-B” in this figure) after 1year of storage with CHOK1-GLP1R cells and cAMP-glo (n=2).

FIG. 5 shows a Western Blot analysis of GLP1-G8_I_VARfeIgG2 (SEQ ID NO:26) (“GLP1B” in this figure) after incubation in serum for 24 hours at37° C. (lane 1), in PBS for 24 hours at 37° C. (lane 2), in PBS for 24hours at 4° C. (lane 9). A mouse anti-GLP1 antibody was used.

FIG. 6 is a plot of GLP1-G8_I_VARfeIgG2 (SEQ ID NO:26) concentration inthe serum over time after subcutaneous administration to 5 cats, asmeasured by quantitative ELISA.

FIG. 7 shows a plot of GLP1-G8_I_VARfeIgG2 (SEQ ID NO:26) concentrationin the serum over time after subcutaneous administration to 5 cats, asmeasured by cell-based activity assay. The mean AUC from 0 to 168 hourswas about 840 μg(h)/mL and the mean t_(1/2) was 36 hours.

DESCRIPTION OF THE SEQUENCES

TABLE 1Table 1 provides a listing of exemplary sequences referenced herein.Description of the Sequences SEQ ID NO: SEQUENCE DESCRIPTION 1PVPEPLGGPSVLIFPPKPKDILRITRIPEVIC Exemplary wild-type canineVVLDLGREDPEVQISWFVDGKEVHTAKTQSRE IgG-A FcQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNH Protein A-IDLPSPIERTISKARGRAHKPSVYVLPPSPKE C1q- LSSSDTVSITCLIKDFYPPDIDVEWQSNGQQECD16- PERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQ QGDPFTCAVMHETLQNHYTDLSLSHSPGK 2PAPEMLGGPSVFIFPPKPKDILLIARTPEVIC Exemplary wild-type canineVVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Protein A+KALPSPIERTISKARGQAHQPSVYVLPPSREE C1q+ LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPCD16+ ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQESLSHSPGK 107PKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPP Exemplary wild-type canineKPKDILLIARTPEVICVVVDLDPEDPEVQISW IgG-B Fc with hingeFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIG Protein A+HQDWLKGKQFTCKVNNKALPSPIERTISKARG C1q+ QAHQPSVYVLPPSREELSKNIVSLICLIKDFFCD16+ PPDIDVEWQSNGQQEPESKYRTTPPQLDEDGS YFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK 3 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCExemplary wild-type canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Protein A-KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPCD16+ ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQISLSHSPGK 108AKECECKCNCNNCPCPGCGLLGGPSVFIFPPK Exemplary wild-type caninePKDILVTARTPTVTCVVVDLDPENPEVQISWF IgG-C Fc with hingeVDSKQVQTANTQPREEQSNGTYRVVSVLPIGH Protein A-QDWLSGKQFKCKVNNKALPSPIEEIISKTPGQ C1q+ AHQPNVYVLPPSRDEMSKNIVTLICLVKDFFPCD16+ PEIDVEWQSNGQQEPESKYRMTPPQLDEDGSY FLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK 4 PVPESLGGPSVFIFPPKPKDILRITRIPEITCExemplary wild-type canine VVLDLGREDPEVQISWFVDGKEVHTAKTQPRE IgG-D FcQQFNSTYRVVSVLPIEHQDWLIGKEFKCRVNH Protein A-IGLPSPIERTISKARGQAHQPSVYVLPPSPKE C1q- LSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPECD16- PESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQ QGDTFTCAVMHEALQNHYTDLSLSHSPGK 5PVPEPLGGPSVLIFPPKPKD T L L I A RTPEVIC Exemplary variant canineVVLDLGREDPEVQISWFVDGKEVHIAKTQSRE IgG-A Fc QQFNGTYRVVSVLPI GHQDWLIGKEFKCRVNH C1q- IDLPSPIERTISKARGRAHKPSVYVLPPSPKE Protein A+LSSSDTVSITCLIKDFYPPDIDVEWQSNGQQE I(21)T PERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQR(23)L QGDPFTCAVMHE A L H NHYTDLSLSHSPGK T(25)A E(80)G T(205)A Q(207)H 6PGCGLLGGPSVFIFPPKPKD T L LI ARTPTVTC Exemplary variant canineVVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN C1q+ KALPSPIEEIISKTPGQAHQPNVYVLPPSRDEProtein A+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP I(21)TESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR V(23)L GDTFICAVMHEALHNHYTQISLSHSPGKT(24)I 7 PVPESLGGPSVFIFPPKPKD T L L I A RTPEITC Exemplary variant canineVVLDLGREDPEVQISWFVDGKEVHTAKTQPRE IgG-D Fc QQFNSTYRVVSVLPI GHQDWLTGKEFKCRVNH C1q- IGLPSPIERTISKARGQAHQPSVYVLPPSPKE Protein A+LSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPE I(21)T PESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQR(23)L QGDTFTCAVMHEAL H NHYTDLSLSHSPGK T(25)A E(80)G Q(207)H 8PVPEPLGGPSVLIFPPKPKDILRITRIPEVIC Exemplary variant canineVVLDLGREDPEVQISWFVDGKEVHTAKTQSRE IgG-A FcQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNH Heterodimer chain 1 T(138)YIDLPSPIERTISKARGRAHKPSVYVLPPSPKE LSSSDTVSI Y CLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQ QGDPFTCAVMHETLQNHYTDLSLSHSPGK 9PVPEPLGGPSVLIFPPKPKDILRITRIPEVIC Exemplary variant canineVVLDLGREDPEVQISWFVDGKEVHTAKTQSRE IgG-A FcQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNH Heterodimer chain 2IDLPSPIERTISKARGRAHKPSVYVLPPSPKE Y(181)TLSSSDTVSITCLIKDFYPPDIDVEWQSNGQQE PERKHRMTPPQLDEDGSYFL T SKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK 10 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary variant canine VVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Heterodimer chain 1 T(137)YKALPSPIERTISKARGQAHQPSVYVLPPSREE LSKNTVSL Y CLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQESLSHSPGK 11PAPEMLGGPSVFIFPPKPKDILLIARTPEVIC Exemplary variant canineVVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Heterodimer chain 2KALPSPIERTISKARGQAHQPSVYVLPPSREE Y(180)TLSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP ESKYRTTPPQLDEDGSYFL T SKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK 12 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCExemplary variant canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Heterodimer chain 1 T(137)YKALPSPIEEIISKTPGQAHQPNVYVLPPSRDE MSKNTVTL Y CLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQISLSHSPGK 13PGCGLLGGPSVFIFPPKPKDILVTARTPTVTC Exemplary variant canineVVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Heterodimer chain 2KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE Y(180)TMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP ESKYRMTPPQLDEDGSYFL T SKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK 14 PVPESLGGPSVFIFPPKPKDILRITRITEITCExemplary variant canine VVLDLGREDPEVQISWFVDGKEVHTAKTQPRE IgG-D FcQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNH Heterodimer chain 1 T(138)YIGLPSPIERTISKARGQAHQPSVYVLPPSPKE LSSSDTVTL Y CLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQ QGDTFTCAVMHEALQNHYTDLSLSHSPGK 15PVPESLGGPSVFIFPPKPKDILRITRITEITC Exemplary variant canineVVLDLGREDPEVQISWFVDGKEVHTAKTQPRE IgG-D FcQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNH Heterodimer chain 2IGLPSPIERTISKARGQAHQPSVYVLPPSPKE Y(181)TLSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPE PESKYHTTAPQLDEDGSYFL T SKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK 109 PVPEPLGGPSVLIFPPKPKDILRITRTPEVICExemplary variant canine VVLDLGREDPEVQISWFVDGKEVHTAKTQSRE IgG-A FcQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNH Heterodimer chain 3IDLPSPIERTISKARGRAHKPSVYVLPPSPKE T(138)W LSSSDTVSI WCLIKDFYPPDIDVEWQSNGQQE PERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK 110 PVPEPLGGPSVLIFPPKPKDILRITRITEVICExemplary variant canine VVLDLGREDPEVQISWFVDGKEVHTAKTQSRE IgG-A FcQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNH Heterodimer chain 4IDLPSPIERTISKARGRAHKPSVYVLPPSPKE T(138)S LSSSDTVSI S C AIKDFYPPDIDVEWQSNGQQE L(140)A PERKHRMTPPQLDEDGSYFL T SKLSVDKSRWQ Y(181)TQGDPFTCAVMHETLQNHYTDLSLSHSPGK 111 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary variant canine VVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Heterodimer chain 3KALPSPIERTISKARGQAHQPSVYVLPPSREE T(137)W LSKNTVSL WCLIKDFFPPDIDVEWQSNGQQEP ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK 112 PAPEMLGGPSVFIFPPKPKDTLLIARTPEVICExemplary variant canine VVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Heterodimer chain 4KALPSPIERTISKARGQAHQPSVYVLPPSREE T(137)S LSKNTVSL S C AIKDFFPPDIDVEWQSNGQQEP L(139)A ESKYRTTPPQLDEDGSYFL T SKLSVDKSRWQR Y(180)TGDTFICAVMHEALHNHYTQESLSHSPGK 113 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCExemplary variant canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Heterodimer chain 3KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE T(137)W MSKNTVTL WCLVKDFFPPEIDVEWQSNGQQEP ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK 114 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCExemplary variant canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Heterodimer chain 4KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE T(137)S MSKNTVTL S C AVKDFFPPEIDVEWQSNGQQEP L(139)A ESKYRMTPPQLDEDGSYFL T SKLSVDKSRWQR Y(180)TGDTFICAVMHEALHNHYTQISLSHSPGK 115 PVPESLGGPSVFIFPPKPKDILRITRITEITCExemplary variant canine VVLDLGREDPEVQISWFVDGKEVHTAKTQPRE IgG-D FcQQFNSTYRVVSVLPIEHQDWLIGKEEKCRVNH Heterodimer chain 3IGLPSPIERTISKARGQAHQPSVYVLPPSPKE T(138)W LSSSDTVTL WCLIKDFFPPEIDVEWQSNGQPE PESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK 116 PVPESLGGPSVFIFPPKPKDILRITRITEITCExemplary variant canine VVLDLGREDPEVQISWFVDGKEVHTAKTQPRE IgG-D FcQQFNSTYRVVSVLPIEHQDWLIGKEEKCRVNH Heterodimer chain 4IGLPSPIERTISKARGQAHQPSVYVLPPSPKE T(138)S LSSSDTVTL S C AIKDFFPPEIDVEWQSNGQPE L(140)A PESKYHTTAPQLDEDGSYFL T SKLSVDKSRWQ Y(181)TQGDTFTCAVMHEALQNHYTDLSLSHSPGK 16 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPExemplary wild-type feline PKPKDTLSISRTPEVICLVVDLGPDDSNVQIT IgG2 FcWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPI Protein A+LHQDWLKGKEFKCKVNSKSLPSAMERTISKAK C1q- GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDG TYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 117 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary wild-type feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1a FcWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPI Protein A+LHQDWLKGKEFKCKVNSKSLPSPIERTISKAK C1q+ GQPHEPQVYVLPPAQEELSENKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDG TYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 118 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary wild-type feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1b FcWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPI Protein A+LHQDWLKGKEFKCKVNSKSLPSPIERTISKDK C1q+ GQPHEPQVYVLPPAQEELSENKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDG TYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 119 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPExemplary variant feline PKPKDTLSISRTPEVICLVVDLGPDDSNVQIT IgG2 FcWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPI Heterodimer chain 1LHQDWLKGKEFKCKVNSKSLPSAMERTISKAK T(154)W GQPHEPQVYVLPPTQEELSENKVSV WCLIKGF HPPDIAVEWEITGQPEPENNYQTTPPQLDSDG TYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 120 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPExemplary variant feline PKPKDTLSISRTPEVICLVVDLGPDDSNVQIT IgG2 FcWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPI Heterodimer chain 2LHQDWLKGKEFKCKVNSKSLPSAMERTISKAK T(154)S GQPHEPQVYVLPPTQEELSENKVSV S C AIKGF L(156)A HPPDIAVEWEITGQPEPENNYQTTPPQLDSDG Y(197)T TYFL TSRLSVDRSHWQRGNTYTCSVSHEALHS HHTQKSLTQSPGK 121RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFP Exemplary variant felinePKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1a FcWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPI Heterodimer chain 1LHQDWLKGKEFKCKVNSKSLPSPIERTISKAK T(154)W GQPHEPQVYVLPPAQEELSENKVSV WCLIKSF HPPDIAVEWEITGQPEPENNYRTTPPQLDSDG TYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 122 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary variant feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1a FcWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPI Heterodimer chain 2LHQDWLKGKEFKCKVNSKSLPSPIERTISKAK T(154)S GQPHEPQVYVLPPAQEELSENKVSV S C AIKSF L(156)A HPPDIAVEWEITGQPEPENNYRTTPPQLDSDG Y(197)T TYFV TSKLSVDRSHWQRGNTYTCSVSHEALHS HHTQKSLTQSPGK 123RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFP Exemplary variant felinePKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1b FcWFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI Heterodimer chain 1LHQDWLKGKEFKCKVNSKSLPSPIERTISKDK T(154)W GQPHEPQVYVLPPAQEELSENKVSV WCLIEGF YPSDIAVEWEITGQPEPENNYRTIPPQLDSDG TYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 124 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary variant feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1b FcWFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI Heterodimer chain 2LHQDWLKGKEFKCKVNSKSLPSPIERTISKDK T(154)S GQPHEPQVYVLPPAQEELSENKVSV S C AIEGF L(156)A YPSDIAVEWEITGQPEPENNYRTTPPQLDSDG Y(197)T TYFL TSRLSVDRSRWQRGNTYTCSVSHEALHS HHTQKSLTQSPGK 17 PKTASTIESKTGE CPKCPVPEIPGAPSVFIFP Exemplary variant felinePKPKDTLSISRTPEVICLVVDLGPDDSNVQIT IgG2 FcWFVDNTEMHTAKTRPREEQENSTYRVVSVLPI Hinge CysLHQDWLKGKEFKCKVNSKSLPSAMERTISKAK G(14)C GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDG TYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 125 RKTDHPPGPKPCDCPKCPPPEMLGGP SVFIFPExemplary variant feline PKPKDTLSISRTPEVTCLVVDLGPDDSNVQITIgG2 Fc with feline IgG1 WFVDNTEMHTAKTRPREEQENSTYRVVSVLPI hingeLHQDWLKGKEFKCKVNSKSLPSAMERTISKAK GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDG TYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 126 PKTASTIESKTGEGP P CPVPEIPGAPSVFIFPExemplary variant feline PKPKDTLSISRTPEVTCLVVDLGPDDSNVQITIgG2 Fc with modified hinge WFVDNTEMHTAKTRPREEQENSTYRVVSVLPI K(16)PLHQDWLKGKEFKCKVNSKSLPSAMERTISKAK GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDG TYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 127 RKTDHPPGPKPCDCP P CPPPEMLGGPSIFIFPExemplary variant feline PKPKDTLSISRTPEVTCLVVDLGPDDSDVQITIgG1a Fc with modified hinge WFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI K(16)PLHQDWLKGKEFKCKVNSKSLPSPIERTISKAK GQPHEPQVYVLPPAQEELSENKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTIPPQLDSDG TYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 128 RKTDHPPGPKPCDCP P CPPPEMLGGPSIFIFPExemplary variant feline PKPKDTLSISRTPEVTCLVVDLGPDDSDVQITIgG1b Fc with modified WFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI hingeLHQDWLKGKEFKCKVNSKSLPSPIERTISKDK K(16)P GQPHEPQVYVLPPAQEELSENKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDG TYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 18 DMSKCPKCPAPELL GGPSVFIFPPNPKDALMIExemplary variant equine Fc SRTPVVTCVVVNLSDQYPDVQFSWYVDNTEVHIgG2 (with equine IgG1 hinge) SAITKQREAQFNSTYRVVSVLPIQHQDWLSGKProtein A+ EFKCSVTNVGVPQPISRAISRGKGPSRVPQVY C1q-VLPPHPDELAKSKVSVTCLVKDFYPPDISVEW QSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFICAVMHEALHNHFIKTDISE SLGK 19 DMSKCPKCPAPELL GGPSVFIFPPNPKD TLMI Exemplary variant equine SRTPVVTCVVVNLSDQYPDVQFSWYVDNTEVHIgG2 Fc (with equine IgG1 hinge) SAITKQREAQFNSTYRVVSVLPIQHQDWLSGK C1q-EFKCSVTNVGVPQPISRAISRGKGPSRVPQVY Protein A+VLPPHPDELAKSKVSVTCLVKDFYPPDISVEW A(29)T QSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSF(217)Y LETSRWQQVESFICAVMHEALHNH Y TKTDISE SLGK 20 H XEGTFTSDVSSYLEGQAAKEFIAWLVKG Exemplary variant GLP1 (7-35)X8 may be G or S 21 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT Glucagon (Gluc) 22HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPS Extendin-4 SGAPPPS 23 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGGG GLP1-G8/GLP-2G_III_SGGGGSGGGGSGGGGSPKTASTIESKTGEGPK WTfeIgG2CPVPEIPGAPSVFIFPPKPKDTLSISRTPEVT CLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPE PENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGKGGG GSGGGGHAEGTFTSDVSSYLEGQAAKEFIAWL VKGGG24 H G EGTFTSDVSSYLEGQAAKEFIAWLVKGGGG GLP1-G8_I_WTfeIgG2SGGGGSGGGGSGGGGSPKTASTIESKTGEGPK CPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPR EEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQE ELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQ RGNTYTCSVSHEALHSHHTQKSLTQSPGK 25 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGGG GLP1-G8/GLP1-2G_III_SGGGGSGGGGSGGGGSPKTASTIESKTGE C PK VARfeIgG2CPVPEIPGAPSVFIFPPKPKDTLSISRTPEVT CLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPE PENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYICSVSHEALHSHHTQKSLTQSPGKGGG GSGGGGHAEGTFTSDVSSYLEGQAAKEFIAWL VKGGG26 H G EGTFTSDVSSYLEGQAAKEFIAWLVKGGGG GLP1-G8_I_VARfeIgG2SGGGGSGGGGSGGGGSPKTASTIESKTGE C PK CPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPR EEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQE ELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQ RGNTYTCSVSHEALHSHHTQKSLTQSPGK 27 H SEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-S8/GLP1-3G_III_GGGGSGGGSGGGSGGGSPKTASTIESKTGEGP WTfeIgG2KCPVPEIPGAPSVFIFPPKPKDTLSISRTPEV TCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKV NSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQP EPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGKGG GGSGGGGHAEGTFTSDVSSYLEGQAAKEFTAWLVKGGGG 28 H S EGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-S8_I_WTfeIgG2GGGGSGGGSGGGSGGGSPKTASTIESKTGEGP KCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRP REEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQ EELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHW QRGNTYTCSVSHEALHSHHTQKSLTQSPGK 29 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8/GLP1-3G_III_GGGGSGGGSGGGSGGGSPKESTSKCISPCPVP VARcaIgGD ESLGGPSVFIFPPKPKD T L L I ARTPEITCVVL DLGREDPEVQISWFVDGKEVHTAKTQPREQQF NSTYRVVSVLPI GHQDWLIGKEEKCRVNHIGL PSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPES KYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEAL H NHYTDLSLSHSPGKGGGGSG GGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKG GGG30 H G EGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8_I_VARcaIgGDGGGGSGGGSGGGSGGGSPKESTCKCISPCPVP ESLGGPSVFIFPPKPKD T L L I A RTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQF NSTYRVVSVLPI G HQDWLIGKEEKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSS SDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGD TFTCAVMHEAL H NHYTDLSLSHSPGK 31 H SEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-88/GLP1-3G_III_GGGGSGGGSGGGSGGGSPKESTSKCISPCPVP VARcaIgGD ESLGGPSVFIFPPKPKD T L L I ARTPEITCVVL DLGREDPEVQISWFVDGKEVHTAKTQPREQQF NSTYRVVSVLPI GHQDWLTGKEFKCRVNHIGL PSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPES KYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEAL H NHYTDLSLSHSPGKGGGGSG GGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKG GGG32 H S EGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-S8_I_VARcaIgGDGGGGSGGGSGGGSGGGSPKESTCKCISPCPVP ESLGGPSVFIFPPKPKD T L L I A RTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQF NSTYRVVSVLPI G HQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSS SDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGD TFTCAVMHEAL H NHYTDLSLSHSPGK 33 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8/GLPL1-3G_III_GGGGSGGGSGGGSGGGSDMSKCPKCPAPELLG VAReqIgG2 GPSVFIFPPNPKD TLMISRTPVVICVVVNLSD QYPDVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPI SRAISRGKGPSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPPDISVEWQSNRWPELEGKYSTT PAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNH Y TKTDISESLGKGGGGSGGGGHA EGTFTSDVSSYLEGQAAKEFIAWLVKGGGG 34 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8_I_VAReqIgG2GGGGSGGGSGGGSGGGSDMSKCPKCPAPELLG GPSVFIFPPNPKD T LMISRTPVVICVVVNLSDQYPDVQFSWYVDNTEVHSAITKQREAQFNSTY RVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPISRAISRGKGPSRVPQVYVLPPHPDELAKSKVS VTCLVKDFYPPDISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFTCA VMHEALHNHYTKTDISESLGK 35 H SEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-88/GLP1-3G_III_GGGGSGGGSGGGSGGGSDMSKCPKCPAPELLG VAReqIgG2 GPSVFIFPPNPKD TLMISRTPVVICVVVNLSD QYPDVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPI SRAISRGKGPSRVPQVYVLPPHPDELAKSKVSVICLVKDFYPPDISVEWQSNRWPELEGKYSIT PAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNH Y TKTDISESLGKGGGGSGGGGHA EGTFTSDVSSYLEGQAAKEFIAWLVKGGGG 36 H SEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-S8_I_VAReqIgG2GGGGSGGGSGGGSGGGSDMSKCPKCPAPELLG GPSVFIFPPNPKD T LMISRTPVVTCVVVNLSDQYPDVQFSWYVDNTEVHSAITKQREAQFNSTY RVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPISRAISRGKGPSRVPQVYVLPPHPDELAKSKVS VTCLVKDFYPPDISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFICA VMHEALHNH Y TKTDISESLGK 37MAVLGLLFCLVTFPSCVLS H S EGTFTSDVSSY ssGLP1-S8/GLP1-3G_III_LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGS WTfeIgG2GGGSPKTASTIESKTGEGPKCPVPEIPGAPSV FIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVS VLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCL IKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHE ALHSHHTQKSLTQSPGKGGGGSGGGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKGGGG 38 MAVLGLLFCLVTFPSCVLS H G EGTFTSDVSSYssGLP1-G8/GLP1-2G_III_ LEGQAAKEFIAWLVKGGGGSGGGGSGGGGSGG VARfeIgG2GGSPKTASTIESKTGE C PKCPVPEIPGAPSVF IFPPKPKDTLSISRTPEVICLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSV LPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLI KGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEA LHSHHTQKSLTQSPGKGGGGSGGGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKGGG 39 MAVLGLLFCLVTFPSCVLS H G EGTFTSDVSSYssGLP1-G8_I_VARfeIgG2 LEGQAAKEFIAWLVKGGGGSGGGGSGGGGSGG GGSPKTASTIESKTGEC PKCPVPEIPGAPSVF IFPPKPKDTLSISRTPEVICLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSV LPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLI KGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEA LHSHHTQKSLTQSPGK 40 MAVLGLLFCLVTFPSCVLSH S EGTFTSDVSSY ssGLP1-S8_I_WTfeIgG2 LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGSGGGSPKTASTIESKTGEGPKCPVPEIPGAPSV FIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVS VLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCL IKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHE ALHSHHTQKSLTQSPGK 41MAVLGLLFCLVTFPSCVLS H G EGIFTSDVSSY ssGLP1-G8/GLP1-2G_III_LEGQAAKEFIAWLVKGGGGSGGGGSGGGGSGG VARcaIgGDGGSPKESTCKCISPCPVPESLGGPSVFIFPPK PKD T L L I A RTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPI G H QDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFF PPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEAL H NH YTDLSLSHSPGKGGGGSGGGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKGGG 42 MAVLGLLFCLVTFPSCVLS H G EGIFTSDVSSYssGLP1-G8/GLP1-2G_III_ LEGQAAKEFIAWLVKGGGGSGGGGSGGGGSGG VARcaIgGDGGSPKEST S KCISPCPVPESLGGPSVFIFPPK PKD T L L I ARTPEITCVVLDLGREDPEVQISWF VDGKEVHTAKTQPREQQFNSTYRVVSVLPI G HQDWLTGKEFKCRVNHIGLPSPIERTISKARGQ AHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGS YFLYSKLSVDKSRWQQGDTFTCAVMHEAL H NHYTDLSLSHSPGKGGGGSGGGGHAEGTFTSDVS SYLEGQAAKEFIAWLVKGGG 43MAVLGLLFCLVTFPSCVLS H S EGTFTSDVSSY ssGLP1-S8/GLP1-3G_III_LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGS VARcaIgGD GGGSPKEST SKCISPCPVPESLGGPSVFIFPP KPKD T L L I A RTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPI G HQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDF FPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEAL H N HYTDLSLSHSPGKGGGGSGGGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKGGGG 44 MAVLGLLFCLVTFPSCVLS H G EGIFTSDVSSYssGLP1-G8/GLP1-3G_III_ LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGS VAReqIgG2GGGSDMSKCPKCPAPELLGGPSVFIFPPNPKD T LMISRIPVVICVVVNLSDQYPDVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVLPIQHQDW LSGKEFKCSVTNVGVPQPISRAISRGKGPSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPPDI SVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNH Y TKT DISESLGKGGGGSGGGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKGGGG 45 MAVLGLLFCLVTFPSCVLS H G EGIFTSDVSSYssGLP1-G8_I_VAReqIgG2 LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGSGGGSSVPKPQCPPYTHSKFLGGPSVFIFPPNP KD T LMISRTPVVICVVVNLSDQYPDVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVLPIQHQ DWLSGKEFKCSVTNVGVPQPISRAISRGKGPSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPP DISVEWQSNRWPELEGKYSITPAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNH Y T KTDISESLGK 46 MAVLGLLFCLVTFPSCVLS H SEGTFTSDVSSY ssGLP1-S8/GLP1-3G_III_ LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGSVAReqIgG2 GGGSDMSKCPKCPAPELLGGPSVFIFPPNPKD TLMISRIPVVICVVVNLSDQYPDVQFSWYVDN TEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPISRAISRGKGPSRV PQVYVLPPHPDELAKSKVSVICLVKDFYPPDISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLY SKLSLETSRWQQVESFTCAVMHEALHNH Y TKTDISESLGKGGGGSGGGGHAEGTFTSDVSSYLE GQAAKEFIAWLVKGGGG 47MAVLGLLFCLVTFPSCVLS H S EGIFTSDVSSY ssGLP1-S8_I_VAReqIgG2LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGS GGGSSVPKPQCPPYTHSKFLGGPSVFIFPPNP KD TLMISRTPVVICVVVNLSDQYPDVQFSWYV DNTEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPISRAISRGKGPS RVPQVYVLPPHPDELAKSKVSVICLVKDFYPPDISVEWQSNRWPELEGKYSITPAQLDGDGSYF LYSKLSLETSRWQQVESFTCAVMHEALHNH Y TKTDISESLGK 48 MGLVAPVVLLHQDDEEHGQDEGPEDGSGYLLGFeline glucagon-like peptide TLTRFSSDFDSAPEVILAPDDQLQLPHPSSRE1 receptor (GLP1R) NFWARTGLCAESFLLRPVGPVGPVMGWSEGFHKRNSRQEFLRRRLFAGGLCAASTQESRNRCSS RGCKSSPADCPELDRTQHLGNSVGPIQAAHQELALGAGGPGDECQCCSVSNSLFIPEPQSTCPY NGYTSWPLEGNLRVACAPPPPPPARTLFGGSRRGAVDKKAGGGNRSPGGGAGTGEFGAPGAGGG LGRRPEVGAWTAAEGTNPADLASSPPPPSTRPPAAPRPPCADFCAASPQTTFPIPSP RRPLPAS GGATVSLSETVQKWREYRHQCQRFLTEAPPPATGLFCNRTFDEYACWPDGLPGSFVNVSCPWYL PWASSVLQGHVYRFCTAEGLWLRQDNSSLPWRNLSECEESKRGERSSPEEQLLSFS IIYTVGYT LSFSALVIASAILLSFRHLHCTRNYIHLNLFASFILRALSVFIRDAVLKWMYSTAPQQHQWDGL LSYQDSLGCRLVFLLMQYCVAANYYWLLVEGVYLYILLAFSVESEQRIFRLYLSIGWGVPLLFV IWGIVKYLYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIK CRLAKSTLTLIPLLGTHEVVFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQ MEFRRSWERWRLKHLHIQRDSSMKPLKCPTSSLTSGGTVGSSVYAASCQASCS 49 RPLPASGGATVSLSETVQKWREYRHQCQRFLTMature feline glucagon-like EAPPPATGLFCNRTFDEYACWPDGLPGSFVNVpeptide 1 receptor-N-terminal SCPWYLPWASSVLQGHVYRFCTAEGLWLRQDNdomain (GLP1R-N) SSLPWRNLSECEESKRGERSSPEEQLLSFS 50 METDTLLLWVLLLWVPGSTGRPLPASGGATVS ssFeGLP1R-N_huFc_ LSETVQKWREYRHQCQRFLTEAPPPATGLFCN PolyHisRTFDEYACWPDGLPGSFVNVSCPWYLPWASSV LQGHVYRFCTAEGLWLRQDNSSLPWRNLSECEESKRGERSSPEEQLLSFSGSENLYFQGPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH 51 METDTLLLWVLLLWVPGSTG RPLPASGGATVS ssFeGLP1R-N_PolyHisLSETVQKWREYRHQCQRFLTEAPPPATGLFCN RTFDEYACWPDGLPGSFVNVSCPWYLPWASSVLQGHVYRFCTAEGLWLRQDNSSLPWRNLSECE ESKRGERSSPEEQLLSFSGGGSHHHHHH 52 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGGG GLP1-G8/Gluc-3G_IV_SGGGGSGGGGSGGGGSPKTASTIESKTGEGPK WTfeIgG2CPVPEIPGAPSVFIFPPKPKDTLSISRTPEVT CLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVN SKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPE PENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYICSVSHEALHSHHTQKSLTQSPGKGGG GSGGGGHSQGTFTSDYSKYLDSRRAQDFVQWL MNTGGG53 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGG Gluc/GLP1-2G_V_GSGGGGSGGGGSGGGGSPKTASTIESKTGEGP WTfeIgG2KCPVPEIPGAPSVFIFPPKPKDTLSISRTPEV TCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKV NSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQP EPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGKGG GGSGGGGHAEGTFTSDVSSYLEGQAAKEFIAW LVKGGG54 H G EGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8/Glu-4G_IV_GGGGSGGGSGGGSGGGSPKEST S KCISPCPVP VARcaIgGD ESLGGPSVFIFPPKPKD T L L I ARTPEITCVVL DLGREDPEVQISWFVDGKEVHTAKTQPREQQF NSTYRVVSVLPI GHQDWLTGKEFKCRVNHIGL PSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPES KYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEAL H NHYTDLSLSHSPGKGGGGSG GGGHSQGTFTSDYSKYLDSRRAQDEVQWLMNT GGGG55 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGAG Gluc/GLP1-3G_V_GGGGSGGGSGGGSGGGSPKEST S KCISPCPVP VARcaIgGD ESLGGPSVFIFPPKPKD T L L I ARTPEITCVVL DLGREDPEVQISWFVDGKEVHTAKTQPREQQF NSTYRVVSVLPI GHQDWLTGKEFKCRVNHIGL PSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPES KYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEAL H NHYTDLSLSHSPGKGGGGSG GGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKG GGG56 H G EGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8/Gluc-4G_IV_GGGGSGGGSGGGSGGGSDMSKCPKCPAPELLG VAReqIgGD GPSVFIFPPNPKD TLMISRTPVVTCVVVNLSD QYPDVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPI SRAISRGKGPSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPPDISVEWQSNRWPELEGKYSTT PAQLDGDGSYFLYSKLSLETSRWQQVESFICAVMHEALHNH Y TKTDISESLGKGGGGSGGGGHS QGTFTSDYSKYLDSRRAQDFVQWLMNTGGGG 57HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGAG Gluc/GLP1-3G_V_GGGGSGGGSGGGSGGGSDMSKCPKCPAPELLG VAReqIgG2 GPSVFIFPPNPKD TLMISRTPVVTCVVVNLSD QYPDVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPI SRAISRGKGPSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPPDISVEWQSNRWPELEGKYSTT PAQLDGDGSYFLYSKLSLETSRWQQVESFICAVMHEALHNH Y TKTDISESLGKGGGGSGGGGHA EGTFTSDVSSYLEGQAAKEFIAWLVKGGGG 58 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8/Glu-4G_IV_GGGGSGGGSGGGSGGGSSESKYGPPCPPCPAP huIgG4 EFLGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGHSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGG G 59 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGGGluc/GLP1-3G_V_huIgG4 GGSGGGSGGGSGGGSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGHA EGTFTSDVSSYLEGQAAKEFIAWLVKGGAGGG G 60PVPEPLGGPSVLIFPPKPKD T LRITRTPEVIC Exemplary variant canineVVLDLGREDPEVQISWFVDGKEVHTAKTQSRE IgG-A FcQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNH C1q- IDLPSPIERTISKARGRAHKPSVYVLPPSPKEProtein A+ LSSSDTVSITCLIKDFYPPDIDVEWQSNGQQE I(21)TPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQ Q(207)H QGDPFTCAVMHETL H NHYTDLSLSHSPGK61 PGCGLLGGPSVFIFPPKPKD T LVTARTPTVTC Exemplary variant canineVVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN C1q+ KALPSPIEEIISKTPGQAHQPNVYVLPPSRDEProtein A+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP I(21)TESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQISLSHSPGK 62PVPESLGGPSVFIFPPKPKD T LRITRTPEITC Exemplary variant canineVVLDLGREDPEVQISWFVDGKEVHTAKTQPRE IgG-D FcQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNH C1q- IGLPSPIERTISKARGQAHQPSVYVLPPSPKEProtein A+ LSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPE I(21)TPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQ Q(207)H QGDTFTCAVMHEAL H NHYTDLSLSHSPGK63 GGPSVFLFPPNPKDILMITRIPEVICVVVDVS Exemplary wild-type equineQENPDVKFNWYMDGVEVRTATTRPKEEQFNST IgG1 FcYRVVSVLRIQHQDWLSGKEFKCKVNNQALPQP Protein A+IERTITKTKGRSQEPQVYVLAPHPDESKKSKV C1q+ SVTCLVKDFYPPEINIEWQSNGQPELETKYSTTQAQQDSDGSYFLYSKLSVDRNRWQQGTTFTC GVMHEALHNHYTQKNVSKNPGK 64GGPSVFIFPPNPKDALMISRTPVVTCVVVNLS Exemplary wild-type equineDQYPDVQFSWYVDNTEVHSAITKQREAQFNST IgG2 FcYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQP Protein A-ISRAISRGKGPSRVPQVYVLPPHPDELAKSKV C1q- SVTCLVKDFYPPDISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFTC AVMHEALHNHFTKTDISESLGK 65GGPSVFIFPPKPKDVLMITRMPEVTCLVVDVS Exemplary wild-type equineHDSSDVLFTWYVDGTEVKTAKTMPNEEQNNST IgG3 FcYRVVSVLRIQHQDWLNGKKFKCKVNNQALPAP Protein A+VERTISKATGQTRVPQVYVLAPHPDELSKNKV C1q+ SVTCLVKDFYPPDITVEWQSNEHPEPEGKYRTTEAQKDSDGSYFLYSKLTVEKDRWQQGTTFTC VVMHEALHNHVMQKNISKNPGK 66VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVG Exemplary wild-type equineHDFPDVQFNWYVDGVETHTATTEPKQEQFNST IgG4 FcYRVVSVLPIQHKDWLSGKEFKCKVNNKALPAP Protein A+VERTISAPTGQPREPQVYVLAPHRDELSKNKV C1q+ SVTCLVKDFYPPDIDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLYSKLTVETNRWQQGTTFTC AVMHEALHNHYTEKSVSKSPGK 67GGPSVFIFPPKPKDVLMISRKPEVTCVVVDLG Exemplary wild-type equineHDDPDVQFTWFVDGVETHTATTEPKEEQFNST IgG5 FcYRVVSVLPIQHQDWLSGKEFKCSVTSKALPAP Protein-VERTISKAKGQLRVPQVYVLAPHPDELAKNTV C1q- SVTCLVKDFYPPEIDVEWQSNEHPEPEGKYSTTPAQLNSDGSYFLYSKLSVETSRWKQGESFTC GVMHEAVENHYTQKNVSHSPGK 68GRPSVFIFPPNPKDTLMISRTPEVTCVVVDVS Exemplary wild-type equineQENPDVKFNWYVDGVEAHTATTKAKEKQDNST IgG6 FcYRVVSVLPIQHQDWRRGKEFKCKVNNRALPAP Protein A-VERTITKAKGELQDPQVYILAPHPDEVTKNTV C1q- SVTCLVKDFYPPDINVEWQSNEEPEPEVKYSTTPAQLDGDGSYFLYSKLTVETDRWEQGESFTC VVMHEAIRHTYRQKSITNFPGK 69VGPSVFIFPPKPKDVLMISRTPTVICVVVDVG Exemplary wild-type equineHDFPDVQFNWYVDGVETHTATTEPKQEQNNST IgG7 FcYRVVSILAIQHKDWLSGKEFKCKVNNQALPAP Protein A+VQKTISKPTGQPREPQVYVLAPHPDELSKNKV C1q+ SVTCLVKDFYPPDIDIEWKSNGQPEPETKYSTTPAQLDGDGSYFLYSKLTVETNRWQQGTTFTC AVMHEALHNHYTEKSVSKSPGK 70GGPSVFLFPPNPKDILMITRIPEVICVVVDVS Exemplary variant equineQENPDVKFNWYMDGVEVRTATTRPKEEQFNST IgG1 Fc YRVVSVLRIQHQDWLSGKEFKC SVNNQALPQP Protein A+ IERTITKTKGRSQEPQVYVLAPHPDESKKSKV C1q-SVTCLVKDFYPPEINIEWQSNGQPELETKYST K(87)S TQAQQDSDGSYFLYSKLSVDRNRWQQGTTFTCGVMHEALHNHYTQKNVSKNPGK 71 GGPSVFIFPPNPKDALMISRTPVVICVVVNLSExemplary variant equine DQYPDVQFSWYVDNTEVHSAITKQREAQFNST IgG2 FcYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQP C1q- ISRAISRGKGPSRVPQVYVLPPHPDELAKSKVProtein A+ SVTCLVKDFYPPDISVEWQSNRWPELEGKYST F(203)YTPAQLDGDGSYFLYSKLSLETSRWQQGESFTC AVMHEALHNH Y TKTDISESLGK 72GGPSVFIFPPNPKD T LMISRTPVVICVVVNLS Exemplary variant equineDQYPDVQFSWYVDNTEVHSAITKQREAQFNST IgG2 FcYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQP C1q- ISRAISRGKGPSRVPQVYVLPPHPDELAKSKVProtein A+ SVTCLVKDFYPPDISVEWQSNRWPELEGKYST A(15)TTPAQLDGDGSYFLYSKLSLETSRWQQVESFTC F(203)Y AVMHEALHNH Y TKTDISESLGK 129PPCVLSAEGVIPIPSVPKPQCPPYTHSKFLGG Exemplary wild-type equinePSVFIFPPNPKDALMISRTPVVTCVVVNLSDQ IgG2 Fc with hingeYPDVQFSWYVDNTEVHSAITKQREAQFNSTYR Protein A-VVSVLPIQHQDWLSGKEFKCSVTNVGVPQPIS C1q- RAISRGKGPSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPPDISVEWQSNRWPELEGKYSTTP AQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNHFTKTDISESLGK 130 PPCVLSAEGVIPIPSVPKPPCPPYTHSKFLGGExemplary variant equine PSVFIFPPNPKDALMISRTPVVTCVVVNLSDQIgG2 Fc with modified hinge YPDVQFSWYVDNTEVHSAITKQREAQFNSTYR Protein A-VVSVLPIQHQDWLSGKEFKCSVTNVGVPQPIS C1q- RAISRGKGPSRVPQVYVLPPHPDELAKSKVSVQ(20)P TCLVKDFYPPDISVEWQSNRWPELEGKYSTTP AQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNHFTKTDISESLGK 131 PPSVLSAEGVIPIPSVPKPQCPPYTHSKFLGGExemplary variant equine PSVFIFPPNPKDALMISRTPVVTCVVVNLSDQIgG2 Fc with modified hinge YPDVQFSWYVDNTEVHSAITKQREAQFNSTYR Protein A-VVSVLPIQHQDWLSGKEFKCSVTNVGVPQPIS C1q- RAISRGKGPSRVPQVYVLPPHPDELAKSKVSVC(3)S TCLVKDFYPPDISVEWQSNRWPELEGKYSTTP AQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNHFTKTDISESLGK 132 PPSVLSAEGVIPIPSVPKPPCPPYTHSKFLGGExemplary variant equine PSVFIFPPNPKDALMISRTPVVTCVVVNLSDQIgG2 Fc with modified hinge YPDVQFSWYVDNTEVHSAITKQREAQFNSTYR Protein A-VVSVLPIQHQDWLSGKEFKCSVTNVGVPQPIS C1q- RAISRGKGPSRVPQVYVLPPHPDELAKSKVSVC(3)S TCLVKDFYPPDISVEWQSNRWPELEGKYSTTP Q(20)PAQLDGDGSYFLYSKLSLETSRWQQVESFICAV MHEALHNHFTKTDISESLGK 133PPCVLSAEGVIPIPSVPKPQCPPYTHSKFLGG Exemplary variant equine PSVFIFPPNPKD TLMISRTPVVICVVVNLSDQ IgG2 Fc with hinge YPDVQFSWYVDNTEVHSAITKQREAQFNSTYRProtein A+ VVSVLPIQHQDWLSGKEFKCSVTNVGVPQPIS C1q-RAISRGKGPSRVPQVYVLPPHPDELAKSKVSV A(45)T TCLVKDFYPPDISVEWQSNRWPELEGKYSTTPF(233)Y AQLDGDGSYFLYSKLSLETSRWQQVESFICAV MHEALHNH Y TKTDISESLGK 134PPCVLSAEGVIPIPSVPKPPCPPYTHSKFLGG Exemplary variant equine PSVFIFPPNPKD TLMISRTPVVTCVVVNLSDQ IgG2 Fc with modified hingeYPDVQFSWYVDNTEVHSAITKQREAQFNSTYR Protein A+VVSVLPIQHQDWLSGKEFKCSVTNVGVPQPIS C1q- RAISRGKGPSRVPQVYVLPPHPDELAKSKVSVQ(20)P TCLVKDFYPPDISVEWQSNRWPELEGKYSTTP A(45)TAQLDGDGSYFLYSKLSLETSRWQQVESFICAV F(233)Y MHEALHNH Y TKTDISESLGK 135PPSVLSAEGVIPIPSVPKPPCPPYTHSKFLGG Exemplary variant equine PSVFIFPPNPKD TLMISRTPVVTCVVVNLSDQ IgG2 Fc with modified hingeYPDVQFSWYVDNTEVHSAITKQREAQFNSTYR Protein A+VVSVLPIQHQDWLSGKEFKCSVTNVGVPQPIS C1q- RAISRGKGPSRVPQVYVLPPHPDELAKSKVSVC(3)S TCLVKDFYPPDISVEWQSNRWPELEGKYSTTP Q(20)PAQLDGDGSYFLYSKLSLETSRWQQVESFICAV A(45)T MHEALHNH Y TKTDISESLGK F(233)Y73 GGPSVFIFPPKPKDVLMITRMPEVTCLVVDVS Exemplary variant equineHDSSDVLFTWYVDGTEVKTAKTMPNEEQNNST IgG3 Fc YRVVSVLRIQHQDWLNGKKFKC SVNNQALPAP Protein A+ VERTISKATGQTRVPQVYVLAPHPDELSKNKV C1q-SVTCLVKDFYPPDITVEWQSNEHPEPEGKYRT K(87)S TEAQKDSDGSYFLYSKLTVEKDRWQQGTTFTCVVMHEALHNHVMQKNISKNPGK 74 VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGExemplary variant equine HDFPDVQFNWYVDGVETHTATTEPKQEQFNST IgG4 FcYRVVSVLPIQHKDWLSGKEFKC S VNNKALPAP Protein A+VERTISAPTGQPREPQVYVLAPHRDELSKNKV C1q- SVTCLVKDFYPPDIDIEWKSNGQPEPETKYSTK(87)S TPAQLDSDGSYFLYSKLTVETNRWQQGTTFTC AVMHEALHNHYTEKSVSKSPGK 75GGPSVFIFPPKPKDVLMISRKPEVTCVVVDLG Exemplary variant equineHDDPDVQFTWFVDGVETHTATTEPKEEQFNST IgG5 FcYRVVSVLPIQHQDWLSGKEFKCSVTSKALPAP C1q- VERTISKAKGQLRVPQVYVLAPHPDELAKNTVProtein A+ SVTCLVKDFYPPEIDVEWQSNEHPEPEGKYST V(199)LTPAQLNSDGSYFLYSKLSVETSRWKQGESFTC E(200)H GVMHEA LH NHYTQKNVSHSPGK 76GRPSVFIFPPNPKDTLMISRTPEVTCVVVDVS Exemplary variant equineQENPDVKFNWYVDGVEAHTATTKAKEKQDNST IgG6 FcYRVVSVLPIQHQDWRRGKEFKCKVNNRALPAP C1q- VERTITKAKGELQDPQVYILAPHPDEVTKNTVProtein A+ SVTCLVKDFYPPDINVEWQSNEEPEPEVKYST I(199)LTPAQLDGDGSYFLYSKLTVETDRWEQGESFTC R(200)H VVMHEA LHNH YRQKSITNFPGKH(201)N T(202)H 77 VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGExemplary variant equine HDFPDVQFNWYVDGVETHTATTEPKQEQNNST IgG7 FcYRVVSILAIQHKDWLSGKEFKC S VNNQALPAP Protein A+VQKTISKPTGQPREPQVYVLAPHPDELSKNKV C1q- SVTCLVKDFYPPDIDIEWKSNGQPEPETKYSTK(87)S TPAQLDGDGSYFLYSKLTVETNRWQQGTTFTC AVMHEALHNHYTEKSVSKSPGK 78PAPEMLGGPSVFIFPPKPKDTLLIARTPEVTC Exemplary variant canineVVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B Fc EQFNGTYRVVSVLPIGHQDWLKGKQFTC RVNN Protein A+ KALPSPIERTISKARGQAHQPSVYVLPPSREE C1q-LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP K(93)R ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK 79 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCExemplary variant canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKC R VNN Protein A-KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q- MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPK(93)R ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQISLSHSPGK 80RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFP Exemplary wild-type felinePKPKDTLSISRTPEVTCLVVDLGPDDSDVQIT IgG1a FcWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPI Protein A+LHQDWLKGKEEKCKVNSKSLPSPIERTISKAK C1q+ GQPHEPQVYVLPPAQEELSENKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTIPPQLDSDG TYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 81 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPExemplary wild-type feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1b FcWFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI Protein A+LHQDWLKGKEEKCKVNSKSLPSPIERTISKDK C1q+ GQPHEPQVYVLPPAQEELSENKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTIPPQLDSDG TYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 136 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary variant feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1a FcWFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI Protein A+ LHQDWLKGKEEKCKVNSKSLPS PIERTISKAK C1q- GQPHEPQVYVLPPAQEELSENKVSVICLIKSF P(198)AHPPDIAVEWEITGQPEPENNYRTIPPQLDSDG TYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 82 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPExemplary variant feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgGla FcWFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI Protein A+ LHQDWLKGKEEKCKVNSKSLPS PIERTISKAK C1q- GQPHEPQVYVLPPAQEELSENKVSVICLIKSF P(198)AHPPDIAVEWEITGQPEPENNYRTIPPQLDSDG TYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 137 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary variant feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1b FcWFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI Protein A+ LHQDWLKGKEEKCKVNSKSLPS PIERTISKDK C1q- GQPHEPQVYVLPPAQEELSENKVSVTCLIEGF P(198)AYPSDIAVEWEITGQPEPENNYRTIPPQLDSDG TYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 83 RKTDHPPGPKTGEGPKCPPPEMLGGPSIFIFPExemplary variant feline PKPKDTLSISRTPEVICLVVDLGPDDSDVQIT IgG1b FcWFVDNIQVYTAKTSPREEQFNSTYRVVSVLPI Protein A+ LHQDWLKGKEEKCKVNSKSLPS PIERTISKDK C1q- GQPHEPQVYVLPPAQEELSENKVSVTCLIEGF P(198)AYPSDIAVEWEITGQPEPENNYRTIPPQLDSDG TYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 84 PGCGLLGGPSVFIFPPKPKD T L LI ARTPTVTCExemplary variant canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQEKC R VNN C1q- KALPSPIEEIISKTPGQAHQPNVYVLPPSRDEK(93)R MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP Protein A+ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR I(21)T GDTFICAVMHEALHNHYTQISLSHSPGKV(23)L T(24)I 85 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGAG Wild-type GLP1 (7-37)86 H S EGTFTSDVSSYLEGQAAKEFIAWLVKG GLP1-S8 (7-35) 87 H GEGTFTSDVSSYLEGQAAKEFIAWLVKG GLP1-G8 (7-35) 88 G 1G extension 89 GG2G extension 90 GGG 3G extension 91 GGGG 4G extension 92 GGGGG5G extension 93 GGGGGG 6G extension 94 GGGGGGG 7G extension 95 GGGGGGGG8G extension 96 MAVLGLLFCLVTFPSCVLS H G EGIFTSDVSSY ssGLP1-G8_I_WTfeIgG2LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGS GGGSPKTASTIESKTGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSN VQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTI SKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQL DSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 97 MAVLGLLFCLVTFPSCVLS H G EGIFTSDVSSYssGLP1-G8/GLP1-3G_III_ LEGQAAKEFIAWLVKGGAGGGGGSGGGSGGGS WTfeIgG2GGGSPKTASTIESKTGEGPKCPVPEIPGAPSV FIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVS VLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCL IKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHE ALHSHHTQKSLTQSPGKGGGGSGGGGHAEGTFTSDVSSYLEGQAAKEFIAWLVKGGGG 98 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGAVariant GLP1 (7-36) 99 HAEGTFTSDVSSYLEGQAAKEFIAWLVKG Variant GLP1 (7-35)100 VPKPQCPPYTHSKFL GGPSVFIFPPNPKDALM Exemplary wild-type equineISRTPVVTCVVVNLSDQYPDVQFSTNYVDNTEV Fc IgG2 (including equineHSAITKQREAQFNSTYRVVSVLPIQHQDWLSG IgG2 hinge)KEFKCSVTNVGVPQPISRAISRGKGPSRVPQV Protein-YVLPPHPDELAKSKVSVTCLVKDFYPPDISVE C1q- WQSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNHFIKTDIS ESLGK 101 DMSKCPKCPAPELLExemplary wild-type equine IgG1 hinge 102 VPKPQCPPYTHSKFLExemplary wild-type equine IgG2 hinge 138 PPCVLSAEGVIPIPSVPKPQCPPYTHSKFLExemplary wild-type equine IgG2 hinge 103 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8/GLP1-3G_III_GGGGSGGGSGGGSGGGSDMSKCPKCPAPELLG VAReqIgG2 GPSVFIFPPNPKD TLMISRTPVVICVVVNLSD QYPDVQFSWYVDNTEVHSAITKQREAQFNSTYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPI SRAISRGKGPSRVPQVYVLPPHPDELAKSKVSVTCLVKDFYPPDISVEWQSNRWPELEGKYSTT PAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNH Y TKTDISESLGKGGGGSGGGGHA EGTFTSDVSSYLEGQAAKEFIAWLVKGGGG 104 HG EGTFTSDVSSYLEGQAAKEFIAWLVKGGAG GLP1-G8_I_VAReqIgG2GGGGSGGGSGGGSGGGSSVPKPQCPPYTHSKF LGGPSVFIFPPNPKD T LMISRTPVVTCVVVNLSDQYPDVQFSWYVDNTEVHSAITKQREAQFNS TYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQPISRAISRGKGPSRVPQVYVLPPHPDELAKSK VSVTCLVKDFYPPDISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFT CAVMHEALHNH Y TKTDISESLGK 105 H GEGTFTSDVSSYLEGQAAKEFIAWLVKGGGG GLP1-G8/GLP1-2G_III_SGGGGSGGGGSGGGGSPKESTCKCISPCPVPE VARcaIgGD SLGGPSVFIFPPKPKD T L L I ARTPEITCVVLD LGREDPEVQISWFVDGKEVHTAKTQPREQQFN STYRVVSVLPI GHQDWLTGKEFKCRVNHIGLP SPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESK YHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEAL H NHYTDLSLSHSPGKGGGGSGG GGHAEGTFTSDVSSYLEGQAAKEFIAWLVKGG G106 H G EGTFTSDVSSYLEGQAAKEFIAWLVKGGGG GLP1-G8/GLP1-2G_III_SGGGGSGGGGSGGGGSPKESTSKCISPCPVPE VARcaIgGD SLGGPSVFIFPPKPKD T L L I ARTPEITCVVLD LGREDPEVQISWFVDGKEVHTAKTQPREQQFN STYRVVSVLPI GHQDWLTGKEFKCRVNHIGLP SPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESK YHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEAL H NHYTDLSLSHSPGKGGGGSGG GGHAEGTFTSDVSSYLEGQAAKEFIAWLVKGG G139 PAPE P LGGPSVFIFPPKPKDTLLTARTPEVIC Exemplary variant canineVVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Protein A+KALPSPIERTISKARGQAHQPSVYVLPPSREE C1q+ LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPCD16- ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQR M(5)PGDTFICAVMHEALHNHYTQESLSHSPGK 140 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary variant canine VVVDLD R EDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Protein A+KALPSPIERTISKARGQAHQPSVYVLPPSREE C1q+ LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPCD16- ESKYRTIPPQLDEDGSYFLYSKLSVDKSRWQR P(39)RGDTFICAVMHEALHNHYTQESLSHSPGK 141 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary variant canine VVVDL G PEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Protein A+KALPSPIERTISKARGQAHQPSVYVLPPSREE C1q+ LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPCD16- ESKYRTIPPQLDEDGSYFLYSKLSVDKSRWQR D(38)GGDTFICAVMHEALHNHYTQESLSHSPGK 142 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary variant canine VVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Protein A+ IALPSPIERTISKARGQAHQPSVYVLPPSREE C1q+ LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPCD16- ESKYRTIPPQLDEDGSYFLYSKLSVDKSRWQR K(97)IGDTFICAVMHEALHNHYTQESLSHSPGK 143 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary variant canine VVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Protein A+ K GLPSPIERTISKARGQAHQPSVYVLPPSREE C1q+ LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPCD16- ESKYRTIPPQLDEDGSYFLYSKLSVDKSRWQR A(98)GGDTFICAVMHEALHNHYTQESLSHSPGK 144 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary variant canine VVVDL G PEDPEVQISWFVDGKQMQTAKTQPRE IgG-B FcEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNN Protein A+ IGLPSPIERTISKARGQAHQPSVYVLPPSREE C1q+ LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPCD16- ESKYRTIPPQLDEDGSYFLYSKLSVDKSRWQR D(38)GGDTFICAVMHEALHNHYTQESLSHSPGK K(97)I A(98)G 145PAPEMLGGPSVFIFPPKPKDILLIARTPEVIC Exemplary variant canine VVVDL GPEDPEVQISWFVDGKQMQTAKTQPRE IgG-B Fc EQFNGTYRVVSVLPIGHQDWLKGKQFTC R VNNProtein A+ IG LPSPIERTISKARGQAHQPSVYVLPPSREE C1q-LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP CD16- ESKYRTIPPQLDEDGSYFLYSKLSVDKSRWQRD(38)G GDTFICAVMHEALHNHYTQESLSHSPGK K(93)R K(97)I A(98)G 146 PAPE PLGGPSVFIFPPKPKDILLIARTPEVIC Exemplary variant canine VVVDLD REDPEVQISWFVDGKQMQTAKTQPRE IgG-B Fc EQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNProtein A+ KALPSPIERTISKARGQAHQPSVYVLPPSREE C1q+LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP CD16- ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRM(5)P GDTFICAVMHEALHNHYTQESLSHSPGK P(39)R 147 PAPE PLGGPSVFIFPPKPKDILLIARTPEVIC Exemplary variant canine VVVDLD REDPEVQISWFVDGKQMQTAKTQPRE IgG-B Fc EQFNGTYRVVSVLPIGHQDWLKGKQFTC R VNNProtein A+ KALPSPIERTISKARGQAHQPSVYVLPPSREE C1q-LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP CD16- ESKYRTIPPQLDEDGSYFLYSKLSVDKSRWQRM(5)P GDTFICAVMHEALHNHYTQESLSHSPGK P(39)R K(93)R 148 PGCG PLGGPSVFIFPPKPKDILVTARTPTVIC Exemplary variant canineVVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Protein A+KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPCD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR L(5)PGDTFICAVMHEALHNHYTQISLSHSPGK 149 PGCGLLGGPSVFIFPPKPKDILVTARTPTVICExemplary variant canine VVVDLD R ENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Protein A+KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPCD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR P(39)RGDTFICAVMHEALHNHYTQISLSHSPGK 150 PGCGLLGGPSVFIFPPKPKDILVTARTPTVICExemplary variant canine VVVDL G PENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Protein A+KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPCD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR D(38)GGDTFICAVMHEALHNHYTQISLSHSPGK 151 PGCGLLGGPSVFIFPPKPKDILVTARTPTVICExemplary variant canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Protein A+ IALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPCD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR K(97)IGDTFICAVMHEALHNHYTQISLSHSPGK 152 PGCGLLGGPSVFIFPPKPKDILVTARTPTVICExemplary variant canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Protein A+ K GLPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPCD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR A(98)GGDTFICAVMHEALHNHYTQISLSHSPGK 153 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCExemplary variant canine VVVDLDPENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKC R VNN Protein A+KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q- MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPCD16+ ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR K(93)RGDTFICAVMHEALHNHYTQISLSHSPGK 154 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCExemplary variant canine VVVDL G PENPEVQISWFVDSKQVQTANTQPRE IgG-C FcEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNN Protein A+ IGLPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q+ MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPCD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR D(38)GGDTFICAVMHEALHNHYTQISLSHSPGK K(97)I A(98)G 155PGCGLLGGPSVFIFPPKPKDILVTARTPTVTC Exemplary variant canine VVVDL GPENPEVQISWFVDSKQVQTANTQPRE IgG-C Fc EQSNGTYRVVSVLPIGHQDWLSGKQFKC R VNNProtein A+ IG LPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q-MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP CD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRD(38)G GDTFICAVMHEALHNHYTQISLSHSPGK K(93)R K(97)I A(98)G 156 PGCG PLGGPSVFIFPPKPKDILVTARTPTVTC Exemplary variant canine VVVDLD RENPEVQISWFVDSKQVQTANTQPRE IgG-C Fc EQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNProtein A+ KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q+MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP CD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRL(5)P GDTFICAVMHEALHNHYTQISLSHSPGK P(39)R 157 PGCG PLGGPSVFIFPPKPKDILVTARTPTVTC Exemplary variant canine VVVDLD RENPEVQISWFVDSKQVQTANTQPRE IgG-C Fc EQSNGTYRVVSVLPIGHQDWLSGKQFKC R VNNProtein A+ KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE C1q-MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP CD16- ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRM(5)P GDTFICAVMHEALHNHYTQISLSHSPGK P(39)R K(93)R 158PVPEPLGGPSVLIFPPKPKDILRITRTPEVIC Exemplary aglycosyl variantVVLDLGREDPEVQISWFVDGKEVHTAKTQSRE canine IgG-A Fc QQFX₁GTYRVVSVLPIEHQDWLIGKEFKCRVNH N(68)X₁ IDLPSPIERTISKARGRAHKPSVYVLPPSPKEX₁ = any amino acid except N LSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQ QGDPFTCAVMHETLQNHYTDLSLSHSPGK 159PVPEPLGGPSVLIFPPKPKDILRITRTPEVIC Exemplary aglycosyl variantVVLDLGREDPEVQISWFVDGKEVHTAKTQSRE canine IgG-A Fc QQFN PTYRVVSVLPIEHQDWLTGKEFKCRVNH G(69)13 IDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQE PERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK 160 PVPEPLGGPSVLIFPPKPKDILRITRTPEVICExemplary aglycosyl variant VVLDLGREDPEVQISWFVDGKEVHTAKTQSREcanine IgG-A Fc QQFNGX ₂YRVVSVLPIEHQDWLTGKEFKCRVNH T(70)X₂IDLPSPIERTISKARGRAHKPSVYVLPPSPKE X₂ = any amino acid except TLSSSDTVSITCLIKDFYPPDIDVEWQSNGQQE or S PERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK 161 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary aglycosyl variant VVVDLDPEDPEVQISWFVDGKQMQTAKTQPREcanine IgG-B Fc EQFX ₁GTYRVVSVLPIGHQDWLKGKQFTCKVNN N(68)X₁KALPSPIERTISKARGQAHQPSVYVLPPSREE X₁ = any amino acid except NLSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP ESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK 162 PAPEMLGGPSVFIFPPKPKDILLIARTPEVICExemplary aglycosyl variant VVVDLDPEDPEVQISWFVDGKQMQTAKTQPREcanine IgG-B Fc EQFN P TYRVVSVLPIGHQDWLKGKQFTCKVNN G(69)13KALPSPIERTISKARGQAHQPSVYVLPPSREE LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQESLSHSPGK 163PAPEMLGGPSVFIFPPKPKDILLIARTPEVIC Exemplary aglycosyl variantVVVDLDPEDPEVQISWFVDGKQMQTAKTQPRE canine IgG-B Fc EQFNGX₂YRVVSVLPIGHQDWLKGKQFTCKVNN T(70)X₂ KALPSPIERTISKARGQAHQPSVYVLPPSREEX₂ = any amino acid except T LSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEP or SESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQESLSHSPGK 164PGCGLLGGPSVFIFPPKPKDILVTARTPTVTC Exemplary aglycosyl variantVVVDLDPENPEVQISWFVDSKQVQTANTQPRE canine IgG-C Fc EQSX₁GTYRVVSVLPIGHQDWLSGKQFKCKVNN N(68)X₁ KALPSPIEEIISKTPGQAHQPNVYVLPPSRDEX₁ = any amino acid except N MSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQR GDTFICAVMHEALHNHYTQISLSHSPGK 165PGCGLLGGPSVFIFPPKPKDILVTARTPTVTC Exemplary aglycosyl variantVVVDLDPENPEVQISWFVDSKQVQTANTQPRE canine IgG-C Fc EQSN PTYRVVSVLPIGHQDWLSGKQFKCKVNN G(69)13 KALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK 166 PGCGLLGGPSVFIFPPKPKDILVTARTPTVTCExemplary aglycosyl variant VVVDLDPENPEVQISWFVDSKQVQTANTQPREcanine IgG-C Fc EQSNGX ₂YRVVSVLPIGHQDWLSGKQFKCKVNN T(70)X₂KALPSPIEEIISKTPGQAHQPNVYVLPPSRDE X₂ = any amino acid except TMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEP or S ESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK 167 PVPESLGGPSVFIFPPKPKDILRITRTPEITCExemplary aglycosyl variant VVLDLGREDPEVQISWFVDGKEVHTAKTQPREcanine IgG-D Fc QQFX ₁STYRVVSVLPIEHQDWLTGKEFKCRVNH N(68)X₁IGLPSPIERTISKARGQAHQPSVYVLPPSPKE X₁ = any amino acid except NLSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPE PESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK 168 PVPESLGGPSVFIFPPKPKDILRITRTPEITCExemplary aglycosyl variant VVLDLGREDPEVQISWFVDGKEVHTAKTQPREcanine IgG-D Fc QQFN P TYRVVSVLPIEHQDWLTGKEFKCRVNH S(69)PIGLPSPIERTISKARGQAHQPSVYVLPPSPKE LSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQ QGDTFTCAVMHEALQNHYTDLSLSHSPGK 169PVPESLGGPSVFIFPPKPKDILRITRTPEITC Exemplary aglycosyl variantVVLDLGREDPEVQISWFVDGKEVHTAKTQPRE canine IgG-D Fc QQFNSX₂YRVVSVLPIEHQDWLTGKEFKCRVNH T(70)X₂ IGLPSPIERTISKARGQAHQPSVYVLPPSPKEX₂ = any amino acid except T LSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPE or SPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQ QGDTFTCAVMHEALQNHYTDLSLSHSPGK 170RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFP Exemplary aglycosyl variantPKPKDTLSISRTPEVTCLVVDLGPDDSDVQIT feline IgG1a Fc WFVDNTQVYTAKTSPREEQFX₁STYRVVSVLPI N(85)X₁ LHQDWLKGKEFKCKVNSKSLPSPIERTISKAKX₁ = any amino acid except N GQPHEPQVYVLPPAQEELSENKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDG TYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 171 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary aglycosyl variant PKPKDTLSISRTPEVTCLVVDLGPDDSDVQITfeline IgG1a Fc WFVDNTQVYTAKTSPREEQFN P TYRVVSVLPI S(86)PLHQDWLKGKEFKCKVNSKSLPSPIERTISKAK GQPHEPQVYVLPPAQEELSENKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDG TYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 172 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary aglycosyl variant PKPKDTLSISRTPEVTCLVVDLGPDDSDVQITfeline IgG1a Fc WFVDNTQVYTAKTSPREEQFNSX ₂YRVVSVLPI T(87)X₂LHQDWLKGKEFKCKVNSKSLPSPIERTISKAK X₂ = any amino acid except TGQPHEPQVYVLPPAQEELSENKVSVTCLIKSF or S HPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHS HHTQKSLTQSPGK 173RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFP Exemplary aglycosyl variantPKPKDTLSISRTPEVTCLVVDLGPDDSDVQIT feline IgG1b Fc WFVDNTQVYTAKTSPREEQFX₁STYRVVSVLPI N(85)X₁ LHQDWLKGKEFKCKVNSKSLPSPIERTISKDKX₁ = any amino acid except N GQPHEPQVYVLPPAQEELSENKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDG TYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 174 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary aglycosyl variant PKPKDTLSISRTPEVTCLVVDLGPDDSDVQITfeline IgG1b Fc WFVDNTQVYTAKTSPREEQFN P TYRVVSVLPI S(86)PLHQDWLKGKEFKCKVNSKSLPSPIERTISKDK GQPHEPQVYVLPPAQEELSENKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDG TYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 175 RKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPExemplary aglycosyl variant PKPKDTLSISRTPEVTCLVVDLGPDDSDVQITfeline IgG1b Fc WFVDNTQVYTAKTSPREEQENSX ₂YRVVSVLPI T(87)X₂LHQDWLKGKEFKCKVNSKSLPSPIERTISKDK X₂ = any amino acid except TGQPHEPQVYVLPPAQEELSENKVSVTCLIEGF or S YPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHS HHTQKSLTQSPGK 176PKTASTIESKTGEGPKCPVPEIPGAPSVFIFP Exemplary aglycosyl variantPKPKDTLSISRTPEVTCLVVDLGPDDSNVQIT feline IgG2 Fc WFVDNTEMHTAKTRPREEQEX₁STYRVVSVLPI N(85)X₁ LHQDWLKGKEFKCKVNSKSLPSAMERTISKAKX₁ = any amino acid except N GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDG TYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 177 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPExemplary aglycosyl variant PKPKDTLSISRTPEVTCLVVDLGPDDSNVQITfeline IgG2 Fc WFVDNTEMHTAKTRPREEQFN P TYRVVSVLPI S(86)PLHQDWLKGKEFKCKVNSKSLPSAMERTISKAK GQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDG TYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK 178 PKTASTIESKTGEGPKCPVPEIPGAPSVFIFPExemplary aglycosyl variant PKPKDTLSISRTPEVTCLVVDLGPDDSNVQITfeline IgG2 Fc WFVDNTEMHTAKTRPREEQENSX ₂YRVVSVLPI T(87)X₂LHQDWLKGKEFKCKVNSKSLPSAMERTISKAK X₂ = any amino acid except TGQPHEPQVYVLPPTQEELSENKVSVTCLIKGF or S HPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHS HHTQKSLTQSPGK 179GGPSVFLEPPNPKDILMITRIPEVICVVVDVS Exemplary aglycosyl variantQENPDVKFNWYMDGVEVRTATTRPKEEQFX ₁ST equine IgG1 FcYRVVSVLRIQHQDWLSGKEFKCKVNNQALPQP N(62)X₁IERTITKTKGRSQEPQVYVLAPHPDESKKSKV X₁ = any amino acid except NSVTCLVKDFYPPEINIEWQSNGQPELETKYST TQAQQDSDGSYFLYSKLSVDRNRWQQGTTFTCGVMHEALHNHYTQKNVSKNPGK 180 GGPSVFLEPPNPKDILMITRIPEVICVVVDVSExemplary aglycosyl variant QENPDVKFNWYMDGVEVRTATTRPKEEQFN P Tequine IgG1 Fc YRVVSVLRIQHQDWLSGKEFKCKVNNQALPQP S(63)PIERTITKTKGRSQEPQVYVLAPHPDESKKSKV SVTCLVKDFYPPEINIEWQSNGQPELETKYSTTQAQQDSDGSYFLYSKLSVDRNRWQQGTTFTC GVMHEALHNHYTQKNVSKNPGK 181GGPSVFLFPPNPKDILMITRIPEVICVVVDVS Exemplary aglycosyl variantQENPDVKFNWYMDGVEVRTATTRPKEEQFNSX ₂ equine IgG1 FcYRVVSVLRIQHQDWLSGKEFKCKVNNQALPQP T(64)X₂IERTITKTKGRSQEPQVYVLAPHPDESKKSKV X₂ = any amino acid except TSVTCLVKDFYPPEINIEWQSNGQPELETKYST or S TQAQQDSDGSYFLYSKLSVDRNRWQQGTTFTCGVMHEALHNHYTQKNVSKNPGK 182 GGPSVFIFPPNPKDALMISRTPVVTCVVVNLSExemplary aglycosyl variant DQYPDVQFSWYVDNTEVHSAITKQREAQFX ₁STequine IgG2 Fc YRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQP N(62)X₁ISRAISRGKGPSRVPQVYVLPPHPDELAKSKV X₁ = any amino acid except NSVTCLVKDFYPPDISVEWQSNRWPELEGKYST TPAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNHFTKTDISESLGK 183 GGPSVFIFPPNPKDALMISRTPVVTCVVVNLSExemplary aglycosyl variant DQYPDVQFSWYVDNTEVHSAITKQREAQFN P Tequine IgG2 Fc YRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQP S(63)PISRAISRGKGPSRVPQVYVLPPHPDELAKSKV SVTCLVKDFYPPDISVEWQSNRWPELEGKYSTTPAQLDGDGSYFLYSKLSLETSRWQQVESFTC AVMHEALHNHFTKTDISESLGK 184GGPSVFIFPPNPKDALMISRTPVVTCVVVNLS Exemplary aglycosyl variantDQYPDVQFSWYVDNTEVHSAITKQREAQFNSX ₂ equine IgG2 FcYRVVSVLPIQHQDWLSGKEFKCSVTNVGVPQP T(64)X₂ISRAISRGKGPSRVPQVYVLPPHPDELAKSKV X₂ = any amino acid except TSVTCLVKDFYPPDISVEWQSNRWPELEGKYST or S TPAQLDGDGSYFLYSKLSLETSRWQQVESFTCAVMHEALHNHFTKTDISESLGK 185 GGPSVFIFPPKPKDVLMITRMPEVTCLVVDVSExemplary aglycosyl variant HDSSDVLFTWYVDGTEVKTAKTMPNEEQNX ₁STequine IgG3 Fc YRVVSVLRIQHQDWLNGKKFKCKVNNQALPAP N(62)X₁VERTISKATGQTRVPQVYVLAPHPDELSKNKV X₁ = any amino acid except NSVTCLVKDFYPPDITVEWQSNEHPEPEGKYRT TEAQKDSDGSYFLYSKLTVEKDRWQQGTTFTCVVMHEALHNHVMQKNISKNPGK 186 GGPSVFIFPPKPKDVLMITRMPEVTCLVVDVSExemplary aglycosyl variant HDSSDVLFTWYVDGTEVKTAKTMPNEEQNN P Tequine IgG3 Fc YRVVSVLRIQHQDWLNGKKFKCKVNNQALPAP S(63)PVERTISKATGQTRVPQVYVLAPHPDELSKNKV SVTCLVKDFYPPDITVEWQSNEHPEPEGKYRTTEAQKDSDGSYFLYSKLTVEKDRWQQGTTFTC VVMHEALHNHVMQKNISKNPGK 187GGPSVFIFPPKPKDVLMITRMPEVTCLVVDVS Exemplary aglycosyl variantHDSSDVLFTWYVDGTEVKTAKTMPNEEQNNSX ₂ equine IgG3 FcYRVVSVLRIQHQDWLNGKKFKCKVNNQALPAP T(64)X₂VERTISKATGQTRVPQVYVLAPHPDELSKNKV X₂ = any amino acid except TSVTCLVKDFYPPDITVEWQSNEHPEPEGKYRT or S TEAQKDSDGSYFLYSKLTVEKDRWQQGTTFTCVVMHEALHNHVMQKNISKNPGK 188 VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGExemplary aglycosyl variant HDFPDVQFNWYVDGVETHTATTEPKQEQFX ₁STequine IgG4 Fc YRVVSVLPIQHKDWLSGKEFKCKVNNKALPAP N(62)X₁VERTISAPTGQPREPQVYVLAPHRDELSKNKV X₁ = any amino acid except NSVTCLVKDFYPPDIDIEWKSNGQPEPETKYST IPAQLDSDGSYFLYSKLTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKSPGK 189 VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGExemplary aglycosyl variant HDFPDVQFNWYVDGVETHTATTEPKQEQFN P Tequine IgG4 Fc YRVVSVLPIQHKDWLSGKEFKCKVNNKALPAP S(63)PVERTISAPTGQPREPQVYVLAPHRDELSKNKV SVTCLVKDFYPPDIDIEWKSNGQPEPETKYSTTPAQLDSDGSYFLYSKLTVETNRWQQGTTFTC AVMHEALHNHYTEKSVSKSPGK 190VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVG Exemplary aglycosyl variantHDFPDVQFNWYVDGVETHTATTEPKQEQFNSX ₂ equine IgG4 FcYRVVSVLPIQHKDWLSGKEFKCKVNNKALPAP T(64)X₂VERTISAPTGQPREPQVYVLAPHRDELSKNKV X₂ = any amino acid except TSVTCLVKDFYPPDIDIEWKSNGQPEPETKYST or S TPAQLDSDGSYFLYSKLTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKSPGK 191 GGPSVFIFPPKPKDVLMISRKPEVTCVVVDLGExemplary aglycosyl variant HDDPDVQFTWFVDGVETHTATTEPKEEQFX ₁PTequine IgG5 Fc YRVVSVLPIQHQDWLSGKEFKCSVTSKALPAP N(62)X₁VERTISKAKGQLRVPQVYVLAPHPDELAKNTV X₁ = any amino acid except NSVTCLVKDFYPPEIDVEWQSNEHPEPEGKYST TPAQLNSDGSYFLYSKLSVETSRWKQGESFTCGVMHEAVENHYTQKNVSHSPGK 192 GGPSVFIFPPKPKDVLMISRKPEVTCVVVDLGExemplary aglycosyl variant HDDPDVQFTWFVDGVETHTATTEPKEEQFN P Tequine IgG5 Fc YRVVSVLPIQHQDWLSGKEFKCSVTSKALPAP S(63)PVERTISKAKGQLRVPQVYVLAPHPDELAKNTV SVTCLVKDFYPPEIDVEWQSNEHPEPEGKYSTTPAQLNSDGSYFLYSKLSVETSRWKQGESFTC GVMHEAVENHYTQKNVSHSPGK 193GGPSVFIFPPKPKDVLMISRKPEVTCVVVDLG Exemplary aglycosyl variantHDDPDVQFTWFVDGVETHTATTEPKEEQFNSX ₂ equine IgG5 FcYRVVSVLPIQHQDWLSGKEFKCSVTSKALPAP T(64)X₂VERTISKAKGQLRVPQVYVLAPHPDELAKNTV X₂ = any amino acid except TSVTCLVKDFYPPEIDVEWQSNEHPEPEGKYST or S TPAQLNSDGSYFLYSKLSVETSRWKQGESFTCGVMHEAVENHYTQKNVSHSPGK 194 GRPSVFIFPPNPKDTLMISRTPEVTCVVVDVSExemplary aglycosyl variant QENPDVKFNWYVDGVEAHTATTKAKEKQDX ₁STequine IgG6 Fc YRVVSVLPIQHQDWRRGKEFKCKVNNRALPAP N(62)X₁VERTITKAKGELQDPQVYILAPHPDEVTKNTV X₁ = any amino acid except NSVTCLVKDFYPPDINVEWQSNEEPEPEVKYST TPAQLDGDGSYFLYSKLTVETDRWEQGESFTCVVMHEAIRHTYRQKSITNFPGK 195 GRPSVFIFPPNPKDTLMISRTPEVTCVVVDVSExemplary aglycosyl variant QENPDVKFNWYVDGVEAHTATTKAKEKQDN P Tequine IgG6 Fc YRVVSVLPIQHQDWRRGKEFKCKVNNRALPAP S(63)PVERTITKAKGELQDPQVYILAPHPDEVTKNTV SVTCLVKDFYPPDINVEWQSNEEPEPEVKYSTIPAQLDGDGSYFLYSKLTVETDRWEQGESFTC VVMHEAIRHTYRQKSITNFPGK 196GRPSVFIFPPNPKDTLMISRTPEVTCVVVDVS Exemplary aglycosyl variantQENPDVKFNWYVDGVEAHTATTKAKEKQDNSX ₂ equine IgG6 FcYRVVSVLPIQHQDWRRGKEFKCKVNNRALPAP T(64)X₂VERTITKAKGELQDPQVYILAPHPDEVTKNTV X₂ = any amino acid except TSVTCLVKDFYPPDINVEWQSNEEPEPEVKYST or S TPAQLDGDGSYFLYSKLTVETDRWEQGESFTCVVMHEAIRHTYRQKSITNFPGK 197 VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGExemplary aglycosyl variant HDFPDVQFNWYVDGVETHTATTEPKQEQNX ₁STequine IgG7 Fc YRVVSILAIQHKDWLSGKEFKCKVNNQALPAP N(62)X₁VQKTISKPTGQPREPQVYVLAPHPDELSKNKV X₁ = any amino acid except NSVTCLVKDFYPPDIDIEWKSNGQPEPETKYST TPAQLDGDGSYFLYSKLTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKSPGK 198 VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVGExemplary aglycosyl variant HDFPDVQFNWYVDGVETHTATTEPKQEQNN P Tequine IgG7 Fc YRVVSILAIQHKDWLSGKEFKCKVNNQALPAP S(63)PVQKTISKPTGQPREPQVYVLAPHPDELSKNKV SVTCLVKDFYPPDIDIEWKSNGQPEPETKYSTTPAQLDGDGSYFLYSKLTVETNRWQQGTTFTC AVMHEALHNHYTEKSVSKSPGK 199VGPSVFIFPPKPKDVLMISRTPTVTCVVVDVG Exemplary aglycosyl variantHDFPDVQFNWYVDGVETHTATTEPKQEQNNSX ₂ equine IgG7 FcYRVVSILAIQHKDWLSGKEFKCKVNNQALPAP T(64)X₂VQKTISKPTGQPREPQVYVLAPHPDELSKNKV X₂ = any amino acid except TSVTCLVKDFYPPDIDIEWKSNGQPEPETKYST or S TPAQLDGDGSYFLYSKLTVETNRWQQGTTFTCAVMHEALHNHYTEKSVSKSPGK

DESCRIPTION OF THE EMBODIMENTS

Variant IgG Fc polypeptides from companion animals, such as canine,equine, and feline, are described. In some embodiments, the variant igGFc polypeptides have increased binding to Protein A, decreased bindingto C1q, decreased binding to CD16, increased stability, increasedrecombinant production, increased hinge disulfide formation, and/or formheterodimeric polypeptides. In some embodiments, antibodies, antibodyfragments, or fusion proteins comprise a variant IgG Fc polypeptide.Methods of producing or purifying variant IgG Fc polypeptides andmethods of administering variant IgG Fc polypeptides to companionanimals are also provided assay.

Also provided are various embodiments relating to contiguouspolypeptides and heterodimeric polypeptides comprising one or morevariant GLP1 polypeptide(s) having improved serum half-life. In someembodiments, the contiguous polypeptides or heterodimeric polypeptidescomprise a GLP1 polypeptide and a glucagon polypeptide as a dual GLP1receptor and glucagon receptor agonist. In some embodiments, suchpolypeptides may be used to treat, for example, diabetes, obesity, orrelated indications, in companion animals, such as canines, felines, andequines.

For the convenience of the reader, the following definitions of termsused herein are provided.

As used herein, numerical terms such as K_(D) are calculated based uponscientific measurements and, thus, are subject to appropriatemeasurement error. In some instances, a numerical term may includenumerical values that are rounded to the nearest significant figure.

As used herein, “a” or “an” means “at least one” or “one or more” unlessotherwise specified. As used herein, the term “or” means “and/or” unlessspecified otherwise. In the context of a multiple dependent claim, theuse of “or” when referring back to other claims refers to those claimsin the alternative only.

Exemplary Variant IgG Fc Polypeptides

Novel variant IgG Fc polypeptides are provided, for example, variant IgGFc polypeptides for increased binding to Protein A, for decreasedbinding to C1q, for decreased binding to CD16, for increased stability,for increased recombinant production, for increased hinge disulfideformation, and/or for forming heterodimeric proteins assay.

“Amino acid sequence,” means a sequence of amino acids residues in apeptide or protein. The terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues, and arenot limited to a minimum length. Such polymers of amino acid residuesmay contain natural or unnatural amino acid residues, and include, butare not limited to, peptides, oligopeptides, dimers, trimers, andmultimers of amino acid residues. Both full-length proteins andfragments thereof are encompassed by the definition. The terms alsoinclude post-expression modifications of the polypeptide, for example,glycosylation, sialylation, acetylation, phosphorylation, and the like.Furthermore, for purposes of the present disclosure, a “polypeptide”refers to a protein which includes modifications, such as deletions,additions, and substitutions (generally conservative in nature), to thenative sequence, as long as the protein maintains the desired activity.These modifications may be deliberate, as through site-directedmutagenesis, or may be accidental, such as through mutations of hostswhich produce the proteins or errors due to PCR amplification.

A “fragment crystallizable polypeptide” or “Fc polypeptide” is theportion of an antibody molecule that interacts with effector moleculesand cells. It comprises the C-terminal portions of the immunoglobulinheavy chains. As used herein, an Fc polypeptide includes fragments ofthe Fc domain having one or more biological activities of an entire Fcpolypeptide. In some embodiments, a biological activity of an Fcpolypeptide is the ability to bind FcRn. In some embodiments, abiological activity of an Fc polypeptide is the ability to bind C1q. Insome embodiments, a biological activity of an Fc polypeptide is theability to bind CD16. In some embodiments, a biological activity of anFc polypeptide is the ability to bind protein A. An “effector function”of the Fc polypeptide is an action or activity performed in whole or inpart by any antibody in response to a stimulus and may includecomplement fixation and/or ADCC (antibody-dependent cellularcytotoxicity) induction.

“IgX Fc” refers to an Fc polypeptide derived from a particular antibodyisotype (e.g., IgG, IgA, IgD, IgE, IgM, etc.), where “X” denotes theantibody isotype. Thus, “IgG Fc” denotes that the Fc polypeptide isderived from a y chain, “IgA Fc” denotes that the Fc polypeptide isderived from an a chain, “IgD Fc” denotes that the Fc polypeptide isderived from a 6 chain, “IgE Fc” denotes that the Fc polypeptide isderived from a c chain, “IgM Fc” denotes that the Fc polypeptide isderived from a μ chain, etc. In some embodiments, the IgG Fc polypeptidecomprises the hinge, CH2, and CH3, but does not comprise CH1 or CL. Insome embodiments, the IgG Fc polypeptide comprises CH2 and CH3, but doesnot comprise CH1, the hinge, or CL. In some embodiments, the IgG Fcpolypeptide comprises CH1, hinge, CH2, CH3, with or without CL. “IgX-NFc” or “IgGXN Fc” denotes that the Fc polypeptide is derived from aparticular subclass of antibody isotype (such as canine IgG subclassIgG-A, IgG-B, IgG-C, or IgG-D; feline IgG subclass IgG1a, IgG1b, orIgG2; or equine IgG subclass IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, orIgG7, etc.), where “N” denotes the subclass.

“Hinge” refers to any portion of an Fc polypeptide or variant Fcpolypeptide that is proline-rich and comprises at least one cysteineresidue located between CH1 and CH2 of a full-length heavy chainconstant region.

In some embodiments, a hinge is capable of forming a disulfide linkagewithin the same hinge region, within the same Fc polypeptide, with ahinge region of a separate Fc polypeptide, or with a separate Fcpolypeptide. In some embodiments, a hinge comprises at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or at least ten prolineresidues.

The term “companion animal species” refers to an animal suitable to be acompanion to humans. In some embodiments, a companion animal species isa canine (or dog), a feline (or cat), or an equine (or horse). In someembodiments, a companion animal species is a small mammal, such as acanine, feline, dog, cat, rabbit, ferret, guinea pig, rodent, etc. Insome embodiments, a companion animal species is a farm animal, such as ahorse, cow, pig, etc.

In some embodiments, an IgX Fc polypeptide or an IgX-N Fc polypeptide isderived from a companion animal, such as a dog, a cat, or a horse. Insome embodiments, IgG Fc polypeptides are isolated from canine y heavychains, such as IgG-A, IgG-B, IgG-C, or IgG-D. In some instances, IgG Fcpolypeptides are isolated from feline y heavy chains, such as IgG1a,IgG1b, or IgG2. In other instances, IgG Fc polypeptides are isolatedfrom equine y heavy chains, such as IgG1, IgG2, IgG3, IgG4, IgG5, IgG6,or IgG7.

The terms “IgX Fc” and “IgX Fc polypeptide” include wild-type IgX Fcpolypeptides and variant IgX Fc polypeptides, unless indicatedotherwise.

“Wild-type” refers to a non-mutated version of a polypeptide that occursin nature, or a fragment thereof. A wild-type polypeptide may beproduced recombinantly.

In some embodiments, a wild-type IgG Fc polypeptide comprises the aminoacid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 16, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 100, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 117,SEQ ID NO: 118.

A “variant” is a polypeptide that differs from a reference polypeptideby single or multiple non-native amino acid substitutions, deletions,and/or additions. In some embodiments, a variant retains at least onebiological activity of the reference polypeptide. In some embodiments, avariant (e.g., a variant canine IgG-A Fc, a variant canine IgG-C Fc, avariant canine IgG-D Fc, variant equine IgG2 Fc, variant equine IgG5 Fc,or variant equine IgG6 Fc) has an activity that the referencepolypeptide substantially lacks. For example, in some embodiments, avariant canine IgG-A Fc, a variant canine IgG-C Fc, a variant canineIgG-D Fc, variant equine IgG2 Fc, variant equine IgG5 Fc, or variantequine IgG6 Fc binds Protein A.

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a nucleic acid molecule or polypeptidesequence are defined as the percentage of nucleotide or amino acidresidues in a reference sequence that are identical with the nucleotideor amino acid residues in the specific nucleic acid molecule orpolypeptide sequence, after aligning the sequences and introducing gaps,if necessary to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software. Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of sequences being compared.

In some embodiments, a variant has at least about 50% sequence identitywith the reference nucleic acid molecule or polypeptide after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Such variants include,for instance, polypeptides wherein one or more amino acid residues areadded, deleted, at the N- or C-terminus of the polypeptide. In someembodiments, a variant has at least about 50% sequence identity, atleast about 60% sequence identity, at least about 65% sequence identity,at least about 70% sequence identity, at least about 75% sequenceidentity, at least about 80% sequence identity, at least about 85%sequence identity, at least about 90% sequence identity, at least about95% sequence identity, at least about 97% sequence identity, at leastabout 98% sequence identity, or at least about 99% sequence identitywith the sequence of the reference nucleic acid or polypeptide.

A “point mutation” is a mutation that involves a single amino acidresidue. The mutation may be the loss of an amino acid, substitution ofone amino acid residue for another, or the insertion of an additionalamino acid residue.

An “amino acid substitution” refers to the replacement of one amino acidin a polypeptide with another amino acid. In some embodiments, an aminoacid substitution is a conservative substitution. Nonlimiting exemplaryconservative amino acid substitutions are shown in Table 2. Amino acidsubstitutions may be introduced into a molecule of interest and theproducts screened for a desired activity, for example, retained/improvedantigen binding, decreased immunogenicity, or improved ADCC or CDC orenhanced pharmacokinetics.

TABLE 2 Original Residue Exemplary Substitutions Ala (A) Val; Leu; IleArg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp; Lys; Arg Asp (D) Glu; AsnCys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H)Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L)Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu;Phe; Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) ThrThr (T) Val; Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V)Ile; Leu; Met; Phe; Ala; Norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions entail exchanging a member of one ofthese classes with another class.

A “variant IgG Fc” as used herein is an IgG Fc polypeptide that differsfrom a reference IgG Fc polypeptide by single or multiple amino acidsubstitutions, deletions, and/or additions and substantially retains atleast one biological activity of the reference IgG Fc polypeptide.

An “amino acid derivative,” as used herein, refers to any amino acid,modified amino acid, and/or amino acid analogue, that is not one of the20 common natural amino acids found in humans. Exemplary amino acidderivatives include natural amino acids not found in humans (e.g.,seleno cysteine and pyrolysine, which may be found in somemicroorganisms) and unnatural amino acids. Exemplary amino acidderivatives, include, but are not limited to, amino acid derivativescommercially available through chemical product manufacturers (e.g.,sigmaaldrich.com/chemistry/chemistry-products.html?TablePage=16274965,accessed on May 6, 2017, which is incorporated herein by reference). Oneor more amino acid derivatives may be incorporated into a polypeptide ata specific location using a translation system that utilizes host cells,orthogonal aminoacyl-tRNA synthetases derived from eubacterialsynthetases, orthogonal tRNAs, and an amino acid derivative. For furtherdescriptions, see, e.g., U.S. Pat. No. 9,624,485.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution with an amino acid derivative. In some embodiments,the amino acid derivative is an alanine derivative, a cysteinederivative, an aspartic acid derivative, a glutamic acid derivative, aphenylalanine derivative, a glycine derivative, a histidine derivative,an isoleucine derivative, a lysine derivative, a leucine derivative, amethionine derivative, an asparagine derivative, a proline derivative, aglutamine derivative, an arginine derivative, a serine derivative, athreonine derivative, a valine derivative, a tryptophan derivative, or atyrosine derivative.

In some embodiments, a variant IgG Fc polypeptide comprises a variantIgG Fc polypeptide of a companion animal species. In some embodiments, avariant IgG Fc polypeptide comprises a variant canine IgG Fcpolypeptide, a variant equine IgG Fc polypeptide, or a feline IgG Fcpolypeptide.

Exemplary Variant IgG Fc Polypeptides with Modified Protein a Binding

In some embodiments, a variant IgG Fc polypeptide has modified Protein Abinding affinity. In some embodiments, a variant IgG Fc polypeptide hasincreased binding affinity to Protein A. In some embodiments, a variantIgG Fc polypeptide may be purified using Protein A columnchromatography.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at a position corresponding to position 21, position23, position 25, position 80, position 205, and/or position 207 of SEQID NO: 1. In some embodiments, a variant IgG Fc polypeptide comprises anamino acid substitution at a position corresponding to position 21,position 23, and/or position 24 of SEQ ID NO: 3. In some embodiments, avariant IgG Fc polypeptide comprises an amino acid substitution at aposition corresponding to position 21, position 23, position 25,position 80, and/or position 207 of SEQ ID NO: 4.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at a position corresponding to position 15, and/orposition 203 of SEQ ID NO: 64. In some embodiments, a variant IgG Fcpolypeptide comprises an amino acid substitution at a positioncorresponding to position 199 and/or position 200 of SEQ ID NO: 67. Insome embodiments, a variant IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 199, position 200,position 201, and/or 202 of SEQ ID NO: 68.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at position 21, position 23, position 25, position 80,position 205, and/or position 207 of SEQ ID NO: 1. In some embodiments,a variant IgG Fc polypeptide comprises an amino acid substitution atposition 21, position 23, and/or position 24 of SEQ ID NO: 3. In someembodiments, a variant IgG Fc polypeptide comprises an amino acidsubstitution at position 21, position 23, position 25, position 80,and/or position 207 of SEQ ID NO: 3.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at position 15 and/or position 203 of SEQ ID NO: 64.In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at position 199 and/or position 200 of SEQ ID NO: 67.In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at position 199, position 200, position 201, and/orposition 202 of SEQ ID NO: 68.

In some embodiments, a variant IgG Fc polypeptide comprises a threonineat a position corresponding to position 21 of SEQ ID NO: 1, a leucine ata position corresponding to position 23 of SEQ ID NO: 1, an alanine at aposition corresponding to position 25 of SEQ ID NO: 1, a glycine at aposition corresponding to position 80 of SEQ ID NO: 1, an alanine at aposition corresponding to position 205 of SEQ ID NO: 1, and/or ahistidine at a position corresponding to position 207 of SEQ ID NO: 1.In some embodiments, a variant IgG Fc polypeptide comprises a threonineat a position corresponding to position 21 of SEQ ID NO: 3, a leucine ata position corresponding to position 23 of SEQ ID NO: 3, and/or anisoleucine at a position corresponding to position 24 of SEQ ID NO: 3.In some embodiments, a variant IgG Fc polypeptide comprises a threonineat a position corresponding to position 21 of SEQ ID NO: 4, a leucine ata position corresponding to position 23 of SEQ ID NO: 4, an alanine at aposition corresponding to position 25 of SEQ ID NO: 4, a glycine at aposition corresponding to position 80 of SEQ ID NO: 3, and/or ahistidine at a position corresponding to position 207 of SEQ ID NO: 4.

In some embodiments, a variant IgG Fc polypeptide comprises a threonineor a valine at a position corresponding to position 15 of SEQ ID NO: 64,and/or a tyrosine or a valine at a position corresponding to position203 of SEQ ID NO: 64. In some embodiments, a variant IgG Fc polypeptidecomprises a leucine at a position corresponding to position 199 of SEQID NO: 67, and/or a histidine at a position corresponding to position200 of SEQ ID NO: 67. In some embodiments, a variant IgG Fc polypeptidecomprises an isoleucine at a position corresponding to position 199 ofSEQ ID NO: 68, a histidine at a position corresponding to position 200of SEQ ID NO: 68, an asparagine at a position corresponding to position201 of SEQ ID NO: 68, and/or a histidine at a position corresponding toposition 202 of SEQ ID NO: 68.

In some embodiments, a variant IgG Fc polypeptide comprises a threonineat position 21 of SEQ ID NO: 1, a leucine at position 23 of SEQ ID NO:1, an alanine at position 25 of SEQ ID NO: 1, a glycine at position 80of SEQ ID NO: 1, an alanine at position 205 of SEQ ID NO: 1, and/or ahistidine at position 207 of SEQ ID NO: 1. In some embodiments, avariant IgG Fc polypeptide comprises a threonine at position 21 of SEQID NO: 3, a leucine at position 23 of SEQ ID NO: 3, and/or an isoleucineat position 24 of SEQ ID NO: 3. In some embodiments, a variant IgG Fcpolypeptide comprise a threonine at a position 21 of SEQ ID NO: 4, aleucine at position 23 of SEQ ID NO: 4, an alanine at position 25 of SEQID NO: 4, a glycine at position 80 of SEQ ID NO: 4, and/or a histidineat position 207 of SEQ ID NO: 4.

In some embodiments, a variant IgG Fc polypeptide comprises a threonineor a valine at position 15 of SEQ ID NO: 64, and/or a tyrosine or avaline at position 203 of SEQ ID NO: 64. In some embodiments, a variantIgG Fc polypeptide comprises a leucine at position 199 of SEQ ID NO: 67,and/or a histidine at position 200 of SEQ ID NO: 67. In someembodiments, a variant IgG Fc polypeptide comprises an isoleucine atposition 199 of SEQ ID NO: 68, a histidine at position 200 of SEQ ID NO:68, an asparagine at position 201 of SEQ ID NO: 68, and/or a histidineat position 202 of SEQ ID NO: 68.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 84. In some embodiments,a variant IgG Fc polypeptide comprises SEQ ID NO: 19, SEQ ID NO: 71, SEQID NO: 72, SEQ ID NO: 75, or SEQ ID NO: 76.

Exemplary Variant IgG Fc Polypeptides with Modified CD16 Binding

In some embodiments, a variant IgG Fc polypeptide has modified CD16binding affinity. In some embodiments, a variant IgG Fc polypeptide hasdecreased binding affinity to CD16. In some embodiments, a variant IgGFc may have a reduced ADCC immune response.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at a position corresponding to position 5, position38, position 39, position 97, and/or position 98 of SEQ ID NO: 2. Insome embodiments, a variant IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 5, position 38,position 39, position 97, and/or position 98 of SEQ ID NO: 3.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at position 5, position 38, position 39, position 97,and/or position 98 of SEQ ID NO: 2. In some embodiments, a variant IgGFc polypeptide comprises an amino acid substitution at position 5,position 38, position 39, position 97, and/or position 98 of SEQ ID NO:3.

In some embodiments, a variant IgG Fc polypeptide comprises a proline ata position corresponding to position 5, a glycine at a positioncorresponding to position 38, an arginine at a position corresponding toposition 39, a isoleucine at a position corresponding to position 97,and/or a glycine at a position corresponding to position 98 of SEQ IDNO: 2. In some embodiments, a variant IgG Fc polypeptide comprises aproline at a position corresponding to position 5, a glycine at aposition corresponding to position 38, an arginine at a positioncorresponding to position 39, a isoleucine at a position correspondingto position 97, and/or a glycine at a position corresponding to position98 of SEQ ID NO: 3.

In some embodiments, a variant IgG Fc polypeptide comprises a proline atposition 5, a glycine at position 38, an arginine at position 39, aisoleucine at position 97, and/or a glycine at position 98 of SEQ ID NO:2. In some embodiments, a variant IgG Fc polypeptide comprises a prolineat position 5, a glycine at position 38, an arginine at position 39, aisoleucine at position 97, and/or a glycine at position 98 of SEQ ID NO:3.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid sequence of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID N: 141, SEQ IDNO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146,SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ IDNO: 151, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156,or SEQ ID NO: 157.

Exemplary Variant IgG Fc Polypeptides with Modified C1q Binding

In some embodiments, a variant IgG Fc polypeptide has modified C1qbinding affinity. In some embodiments, a variant IgG Fc polypeptide hasreduced binding affinity to C1q. In some embodiments, a variant IgG Fcpolypeptide may have reduced complement fixation. In some embodiments, avariant IgG Fc may have a reduced complement-mediated immune response.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at a position corresponding to position 93 of SEQ IDNO: 2. In some embodiments, a variant IgG Fc polypeptide comprises anamino acid substitution at a position corresponding to position 93 ofSEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptidecomprises an amino acid substitution at a position corresponding toposition 87 of SEQ ID NO: 63. In some embodiments, a variant IgG Fcpolypeptide comprises an amino acid substitution at a positioncorresponding to position 87 of SEQ ID NO: 65. In some embodiments, avariant IgG Fc polypeptide comprises an amino acid substitution at aposition corresponding to position 87 of SEQ ID NO: 66. In someembodiments, a variant IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 198 of SEQ ID NO:80. In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at a position corresponding to position 198 of SEQ IDNO: 81.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at position 93 of SEQ ID NO: 2. In some embodiments, avariant IgG Fc polypeptide comprises an amino acid substitution atposition 93 of SEQ ID NO: 3. In some embodiments, a variant IgG Fcpolypeptide comprises an amino acid substitution at position 87 of SEQID NO: 63. In some embodiments, a variant IgG Fc polypeptide comprisesan amino acid substitution at position 87 of SEQ ID NO: 65. In someembodiments, a variant IgG Fc polypeptide comprises or an amino acidsubstitution at position 87 of SEQ ID NO: 66. In some embodiments, avariant IgG Fc polypeptide comprises an amino acid substitution atposition 198 of SEQ ID NO: 80. In some embodiments, a variant IgG Fcpolypeptide comprises an amino acid substitution at position 198 of SEQID NO: 81.

In some embodiments, a variant IgG Fc polypeptide comprises an arginineat a position corresponding to position 93 of SEQ ID NO: 2. In someembodiments, a variant IgG Fc polypeptide comprises an arginine at aposition corresponding to position 93 of SEQ ID NO: 3. In someembodiments, a variant IgG Fc polypeptide comprises a serine at aposition corresponding to position 87 of SEQ ID NO: 63. In someembodiments, a variant IgG Fc polypeptide comprises a serinesubstitution at a position corresponding to position 87 of SEQ ID NO:65. In some embodiments, a variant IgG Fc polypeptide comprises a serineat a position corresponding to position 87 of SEQ ID NO: 66. In someembodiments, a variant IgG Fc polypeptide comprises an alanine at aposition corresponding to position 198 of SEQ ID NO: 80. In someembodiments, a variant IgG Fc polypeptide comprises an alanine at aposition corresponding to position 198 of SEQ ID NO: 81.

In some embodiments, a variant IgG Fc polypeptide comprises an arginineat position 93 of SEQ ID NO: 2. In some embodiments, a variant IgG Fcpolypeptide comprises an amino acid substitution at position 93 of SEQID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises aserine at position 87 of SEQ ID NO: 63. In some embodiments, a variantIgG Fc polypeptide comprises a serine at position 87 of SEQ ID NO: 65.In some embodiments, a variant IgG Fc polypeptide comprises a serine atposition 87 of SEQ ID NO: 66. In some embodiments, a variant IgG Fcpolypeptide comprises an alanine at position 198 of SEQ ID NO: 80. Insome embodiments, a variant IgG Fc polypeptide comprises an alanine atposition 198 of SEQ ID NO: 81.

In some embodiments, a variant IgG Fc polypeptide comprises the aminoacid sequence of SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 84. In someembodiments, a variant IgG Fc polypeptide comprises the amino acidsequence of SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO:77. In some embodiments, a variant IgG Fc polypeptide comprises theamino acid sequence of SEQ ID NO: 82 or SEQ ID NO: 83.

Exemplary Variant IgG Fc Polypeptides with a Modified Inter-ChainDisulfide Linkage

In some embodiments, a variant feline IgG Fc polypeptide has at leastone additional inter-chain disulfide linkage relative to the wild-typefeline IgG Fc polypeptide. In some embodiments, a variant feline IgG Fcpolypeptide has at least one additional inter-chain disulfide linkage inthe hinge region. In some embodiments, a variant feline IgG2 Fcpolypeptide with at least one additional inter-chain disulfide linkagehas increased inter-chain stability relative to the wild-type feline IgGFc polypeptide. In some embodiments, a variant IgG polypeptide has atleast one amino acid modification to a hinge region relative to awild-type IgG Fc polypeptide. In some embodiments, the wild-type IgG Fcpolypeptide is a wild-type feline or equine IgG Fc polypeptide. In someembodiments, the variant IgG Fc polypeptide comprises a hinge region ora portion of a hinge region from an IgG Fc polypeptide of a differentisotype. In some embodiments, the variant IgG Fc polypeptide comprises ahinge region from a wild-type feline IgG-1a Fc polypeptide, from awild-type feline IgG-1b Fc polypeptide, or from a wild-type equine IgG1Fc polypeptide. In some embodiments, a variant IgG2 Fc polypeptide hasincreased recombinant production and/or increased hinge disulfideformation relative to the wild-type IgG Fc polypeptide. In someembodiments, the increased recombinant production and/or increased hingedisulfide formation can be determined by SDS-PAGE analysis underreducing and/or non-reducing conditions.

In some embodiments, a variant IgG Fc polypeptide comprises a cysteineat a position corresponding to position 8, position 9, position 10,position 11, position 12, position 13, position 14, position 15, orposition 16 of SEQ ID NO: 16. In some embodiments, a variant IgG Fcpolypeptide comprises a cysteine at position 8, position 9, position 10,position 11, position 12, position 13, position 14, position 15, orposition 16 of SEQ ID NO: 16. In some embodiments, a variant IgG Fcpolypeptide comprises SEQ ID NO: 17.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at a position corresponding to position 16 of SEQ IDNO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at a position corresponding to position 3 and/or at aposition corresponding to position 20 of SEQ ID NO: 129.

In some embodiments, a variant IgG Fc polypeptide comprises an aminoacid substitution at position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 117, or SEQ ID NO: 118. In some embodiments, avariant IgG Fc polypeptide comprises an amino acid substitution atposition 3 and/or at a position corresponding to position 20 of SEQ IDNO: 129.

In some embodiments, a variant IgG Fc polypeptide comprises a proline ata position corresponding to position 16 of SEQ ID NO: 16, SEQ ID NO: 80,SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118. In some embodiments, avariant IgG Fc polypeptide comprises a serine at a positioncorresponding to position 3 and/or a proline at a position correspondingto position 20 of SEQ ID NO: 129.

In some embodiments, a variant IgG Fc polypeptide comprises a proline atposition 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:117, or SEQ ID NO: 118. In some embodiments, a variant IgG Fcpolypeptide comprises a serine at position 3 and/or a proline atposition 20 of SEQ ID NO: 129.

In some embodiments, the variant IgG Fc polypeptide comprises SEQ ID NO:19, SEQ ID NO: 125 or SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128,SEQ ID NO: SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID N: 134, SEQ ID NO: 135.

Exemplary Variant IgG Fc Polypeptides for Heterodimeric Polypeptides

In certain embodiments, a heterodimeric polypeptide provided herein is aa bispecific antibody. A bispecific antibody has a binding specificityfor two different epitopes or target molecules. In some embodiments, abispecific antibody binds to two different epitopes of the same targetmolecule. Bispecific antibodies may be full length antibodies orantibody fragments.

In some embodiments, the heterodimeric polypeptide comprises a firstvariant IgG Fc polypeptide comprising a “knob” mutation and a secondvariant IgG Fc polypeptide comprising a “hole” mutation. Nonlimitingexemplary knob and hole mutations are described, for example, inMerchant, A. M. et al. An efficient route to human bispecific IgG. NatBiotechnol, 16(7):677-81 (1998).

In some embodiments, a variant canine or variant feline IgG Fcpolypeptide comprises a knob mutation. In some embodiments, a variantIgG Fc polypeptide comprises a tyrosine or a tryptophan at a positioncorresponding to position 138 of SEQ ID NO: 1. In some embodiments, avariant IgG Fc polypeptide comprises a tyrosine or a tryptophan at aposition corresponding to position 137 of SEQ ID NO: 2. In someembodiments, a variant IgG Fc polypeptide comprises a tyrosine or atryptophan at a position corresponding to position 137 of SEQ ID NO: 3.In some embodiments, a variant IgG Fc polypeptide comprises a tyrosineor a tryptophan at a position corresponding to position 138 of SEQ IDNO: 4. In some embodiments, a variant IgG Fc polypeptide comprises atryptophan at a position corresponding to position 154 of SEQ ID NO: 16,SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.

In some embodiments, a variant IgG Fc polypeptide comprises a tyrosineor a tryptophan at position 138 of SEQ ID NO: 1. In some embodiments, avariant IgG Fc polypeptide comprises a tyrosine or a tryptophan atposition 137 of SEQ ID NO: 2. In some embodiments, a variant IgG Fcpolypeptide comprises a tyrosine or a tryptophan at position 137 of SEQID NO: 3. In some embodiments, a variant IgG Fc polypeptide comprises atyrosine or a tryptophan at position 138 of SEQ ID NO: 4. In someembodiments, a variant IgG Fc polypeptide comprises a tryptophan atposition 154 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:117, or SEQ ID NO: 118.

In some embodiments, a variant IgG Fc polypeptide comprising a knobmutation comprises an amino acid sequence of SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQ ID NO: 111, SEQ IDNO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO:123.

In some embodiments, a variant canine or a variant feline IgG Fcpolypeptide comprises a hole mutation. In some embodiments, a variantIgG Fc polypeptide comprises a serine at a position corresponding toposition 138, an alanine at a position corresponding to position 140,and/or a threonine at a position corresponding to position 181 of SEQ IDNO: 1. In some embodiments, a variant IgG Fc polypeptide comprises aserine at a position corresponding to position 137, an alanine at aposition corresponding to position 139, and/or a threonine at a positioncorresponding to position 180 of SEQ ID NO: 2. In some embodiments, avariant IgG Fc polypeptide comprises a serine at a positioncorresponding to position 137, an alanine at a position corresponding toposition 139, and/or a threonine at a position corresponding to position180 of SEQ ID NO: 3. In some embodiments, a variant IgG Fc polypeptidecomprises a serine at a position corresponding to position 138, analanine at a position corresponding to position 140, and/or a threonineat a position corresponding to position 181 of SEQ ID NO: 4. In someembodiments, a variant IgG Fc polypeptide comprises a serine at aposition corresponding to position 154, an alanine at a positioncorresponding to position 156, and/or a threonine at a positioncorresponding to position 197 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.

In some embodiments, a variant IgG Fc polypeptide comprises a serine atposition 138, an alanine at position 140, and/or a threonine at position181 of SEQ ID NO: 1. In some embodiments, a variant IgG Fc polypeptidecomprises a serine at position 137, an alanine at position 139, and/or athreonine at position 181 of SEQ ID NO: 2. In some embodiments, avariant IgG Fc polypeptide comprises a serine at position 137, analanine at position 139, and/or a threonine at position 181 of SEQ IDNO: 3. In some embodiments, a variant IgG Fc polypeptide comprises aserine at position 138, an alanine at position 140, and/or a threonineat position 181 of SEQ ID NO: 4. In some embodiments, a variant IgG Fcpolypeptide comprises a serine at position 154, an alanine at position156, and/or a threonine at position 197 of SEQ ID NO: 16, SEQ ID NO: 80,SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118.

In some embodiments, a variant IgG Fc polypeptide comprising a holemutation comprises an amino acid sequence of SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ IDNO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO:124.

In some embodiments, a contiguous polypeptide comprises a GLP1polypeptide and a variant canine or feline IgG Fc polypeptide comprisinga knob mutation. In some embodiments, a contiguous polypeptide comprisesa glucagon polypeptide and a variant canine or feline IgG Fc polypeptidecomprising a knob mutation. In some embodiments, a contiguouspolypeptide comprises a GLP1 polypeptide and a variant canine or felineIgG Fc polypeptide comprising a hole mutation. In some embodiments, acontiguous polypeptide comprises a glucagon polypeptide and a variantcanine or feline IgG Fc polypeptide comprising a hole mutation.

In some embodiments, the heterodimeric polypeptide comprises a firstcontiguous polypeptide comprises a GLP1 polypeptide and a variant canineor feline IgG Fc polypeptide comprising a knob mutation, and a secondcontiguous polypeptide comprises a glucagon polypeptide and a variantcanine or feline IgG Fc polypeptide comprising a hole mutation. In someembodiments, the heterodimeric polypeptide comprises a first contiguouspolypeptide comprises a glucagon polypeptide and a variant canine orfeline IgG Fc polypeptide comprising a knob mutation, and a secondcontiguous polypeptide comprises a GLP1 polypeptide and a variant canineor feline IgG Fc polypeptide comprising a hole mutation.

Exemplary GLP1 Polypeptides and Glucagon Polypeptides

“GLP1” or a “GLP1 polypeptide,” as used herein, is a polypeptidecomprising the entirety or a fragment of glucagon-like peptide-1 thatbinds to a glucagon-like peptide 1 receptor (GLP1R).

For example, “GLP1” refers to a GLP1 polypeptide from any vertebratesource, including mammals such as primates (e.g., humans and cynomolgusmonkeys), rodents (e.g., mice and rats), and companion animals (e.g.,dogs, cats, and equine), unless otherwise indicated. In someembodiments, a GLP1 polypeptide is a wild-type GLP1 polypeptide, such asGLP1 (7-37) (SEQ ID N: 85). In some embodiments, a GLP1 polypeptide is avariant GLP1 polypeptide, such as GLP1 (7-36) (SEQ ID NO: 98), GLP1(7-35) (SEQ ID NO: 99), GLP1-S8 (7-35) (SEQ ID NO: 86), or GLP1-G8(7-35) (SEQ ID NO: 87). In some embodiments, GLP1 comprises the aminoacid sequence of SEQ ID NO: 20.

“GLP1R,” as used herein, is a polypeptide comprising the entirety or afragment of a glucagon-like peptide 1 receptor that is capable ofbinding to a wild-type GLP1.

For example, “GLP1R” refers to a GLP1R polypeptide from any vertebratesource, including mammals such as primates (e.g., humans and cynomolgusmonkeys), rodents (e.g., mice and rats), and companion animals (e.g.,dogs, cats, and equine), unless otherwise indicated. In someembodiments, GLP1R is an extracellular domain fragment that binds awild-type GLP1 polypeptide. In some embodiments, a feline GLP1Rcomprises the amino acid sequence of SEQ ID NO: 49. In some embodiments,a feline GLP1R comprises the amino acid sequence of SEQ ID NO: 48.

An “extracellular domain” (“ECD”) is the portion of a polypeptide thatextends beyond the transmembrane domain into the extracellular space.The term “extracellular domain,” as used herein, may comprise a completeextracellular domain or may comprise a truncated extracellular domainmissing one or more amino acids, that binds to its ligand. Thecomposition of the extracellular domain may depend on the algorithm usedto determine which amino acids are in the membrane. Different algorithmsmay predict, and different systems may express, different extracellulardomains for a given protein.

“Glucagon” or a “glucagon polypeptide,” as used herein, is a polypeptidecomprising the entirety or a fragment of glucagon that binds to aglucagon receptor.

For example, “glucagon” refers to a glucagon polypeptide from anyvertebrate source, including mammals such as primates (e.g., humans andcynomolgus monkeys), rodents (e.g., mice and rats), and companionanimals (e.g., dogs, cats, and equine), unless otherwise indicated. Insome embodiments, a glucagon polypeptide is a wild-type glucagonpolypeptide, such as SEQ ID NO: 21.

Exemplary Variant IgG Fc Polypeptides and Fusion Molecules

Polypeptides and other molecules may comprise a variant IgG Fcpolypeptide. In some embodiments, a fusion molecule comprises a variantIgG Fc polypeptide, such as the variant IgG Fc polypeptides describedherein. In some embodiments, an antibody or an antibody fragmentcomprises a variant IgG Fc polypeptide, such as the variant IgG Fcpolypeptides described herein.

A “fusion molecule,” as used herein, refers to a molecule comprising oneor more “fusion partners.” In some embodiments, the fusion partners arecovalently linked (“fused”). If two fusion partners are bothpolypeptides, the fusion partner polypeptides may be part of acontiguous amino acid sequence (i.e., a contiguous polypeptide). A firstfusion partner polypeptide may be linked to either the N-terminus or theC-terminus of a second fusion partner. In some embodiments, the fusionpartners are translated as a single polypeptide from a coding sequencethat encodes both fusion partners. Fusion partners may be covalentlylinked through other means, such as, for example, a chemical linkageother than a peptide bond. Many known methods of covalently linkingpolypeptides to other molecules (for example, fusion partners) may beused. In other embodiments, the fusion partners are fused through a“linker,” which is comprised of at least one amino acid or chemicalmoiety. In some embodiments, fusion partners are noncovalently linked.In some such embodiments, they may be linked, for example, using bindingpairs. Exemplary binding pairs include, but are not limited to, biotinand avidin or streptavidin, an antibody and its antigen, etc.

In some embodiments, the fusion partners include an IgG Fc polypeptideand at least one GLP1 polypeptide. In some embodiments, the fusionpartners include an IgG Fc polypeptide, a GLP1 polypeptide, and aglucagon polypeptide. In some embodiments, a GLP1 polypeptide may belinked to either the N-terminus or the C-terminus of an IgG Fcpolypeptide. In some embodiments, a glucagon polypeptide may be linkedto either the N-terminus or the C terminus of an IgG Fc polypeptide.

The term “contiguous polypeptide” herein is used to mean anuninterrupted sequence of amino acids. A contiguous polypeptide istypically translated from a single continuous DNA sequence. It can bemade by genetic engineering, for example, by removing the stop codonfrom the DNA sequence of the first protein, then appending the DNAsequence of the second protein in frame, so that the DNA sequence isexpressed as a single protein. Typically, this is accomplished bycloning a cDNA into an expression vector in frame with an existing gene.

A “linker” refers to one or more amino acid residues that connects afirst polypeptide with a second polypeptide.

In some embodiments, the linker is a flexible, non-structural linker. Insome embodiments, the linker is a glycine-rich, serine-rich, or glycine-and serine-rich linker. In some embodiments, a linker comprises 100%, atleast 95%, at least 90%, or at least 85% serine and/or glycine aminoacid residues.

An “extension,” as used herein, refers to one or more amino acidresidues that are connected to a polypeptide at its C-terminus or at itsN-terminus.

In some embodiments, an extension is flexible. In some embodiments, theextension adds flexibility to the polypeptide without interfering withthe biological activity of the polypeptide. In some embodiments, theextension increases solubility of the polypeptide. In some embodiments,the extension comprises one or more glycine residues. In someembodiments, the extension comprises a glycine residue (SEQ ID NO: 88),two glycine residues (SEQ ID NO: 89), a three glycine residues (SEQ IDNO: 90), four glycine residues (SEQ ID NO: 91), five glycine residues(SEQ ID NO: 92), six glycine residues (SEQ ID NO: 93), seven glycineresidues (SEQ ID NO: 94), eight glycine residues (SEQ ID NO: 95), ormore glycine residues.

In some embodiments, the contiguous polypeptide comprises an IgG Fcpolypeptide comprising an amino acid sequence of any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199and a GLP1 polypeptide comprising an amino acid sequence of SEQ ID NO:85. In some embodiments, the contiguous polypeptide comprises an IgG Fcpolypeptide comprising an amino acid sequence of any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199and a GLP1 polypeptide comprising an amino acid sequence of SEQ ID NO:86. In some embodiments, the contiguous polypeptide comprises an IgG Fcpolypeptide comprising an amino acid sequence of any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199and a GLP1 polypeptide comprising an amino acid sequence of SEQ ID NO:87. In some embodiments, the contiguous polypeptide comprises an IgG Fcpolypeptide comprising an amino acid sequence of any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199and a GLP1 polypeptide comprising an amino acid sequence of SEQ ID NO:98. In some embodiments, the contiguous polypeptide comprises an IgG Fcpolypeptide comprising an amino acid sequence of any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199and a GLP1 polypeptide comprising an amino acid sequence of SEQ ID NO:99.

In some embodiments, the contiguous polypeptide comprises an IgG Fcpolypeptide comprising an amino acid sequence of any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 100, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 167,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199and a glucagon polypeptide comprising an amino acid sequence of SEQ IDNO: 21.

In some embodiments, a contiguous polypeptide comprises a first GLP1polypeptide (GLP1A), a first linker (L1), an Fc polypeptide of acompanion animal species, optionally a second linker (L2), andoptionally a second GLP1 polypeptide (GLP1B). In some embodiments, thecontiguous polypeptide comprises:

GLP1A-L1-Fc;  Formula (I):

Fc-L1-GLP1A; or  Formula (II):

GLP1A-L1-Fc-L2-GLP1B.  Formula (III):

In some embodiments, a contiguous polypeptide comprises a GLP1polypeptide, a first linker (L1), an Fc polypeptide, a second linker(L2), and a glucagon polypeptide (Gluc). In some embodiments, thecontiguous polypeptide comprises:

GLP1-L1-Fc-L2-Gluc; or  Formula (IV):

Gluc-L1-Fc-L2-GLP1.  Formula (V):

In some embodiments, the GLP1 fusion molecule has an increased serumhalf-life compared to a wild-type GLP1 polypeptide. The increasedhalf-life of the GLP1 fusion molecules described herein may requirelower doses and less-frequent dosing regimen than wild-type GLP1polypeptides.

In some embodiments, GLP1B, if present, comprises the same amino acidsequence as GLP1A.

In some embodiments, GLP1, GLP1A, or GLP1B, if present, comprises awild-type GLP1 polypeptide. In some embodiments, GLP1, GLP1A, or GLP1B,if present, comprises a variant GLP1 polypeptide. In some embodiments,GLP1, GLP1A, or GLP1B, if present, comprises an amino acid sequence ofSEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98, or SEQ IDNO: 99.

In some embodiments, the glucagon polypeptide comprises a wild-typeglucagon polypeptide. In some embodiments, the glucagon polypeptidecomprises an amino acid sequence of SEQ ID NO: 21. In some embodiments,the glucagon polypeptide comprises a variant glucagon polypeptide.

In some embodiments, the Fc polypeptide is a human IgG Fc. In someembodiments, the Fc polypeptide is a human IgG1 Fc, a human IgG2 Fc, ahuman IgG3 Fc, or a human IgG4 Fc. In some embodiments, the Fcpolypeptide is a variant human IgG Fc.

In some embodiments, the Fc polypeptide is an IgG Fc from a companionanimal.

In some embodiments, the Fc polypeptide is a canine IgG-A Fc, a canineIgG-B Fc, a canine IgG-C Fc, a canine IgG-D Fc. In some embodiments, theFc is an equine IgG1 Fc, an equine IgG2 Fc, an equine IgG3 Fc, an equineIgG4 Fc, an equine IgG5 Fc, an equine IgG6 Fc, or an equine IgG7 Fc. Insome embodiments, the Fc is a feline IgG1a Fc, a feline IgG1b Fc, or afeline IgG2 Fc.

In some embodiments, the Fc polypeptide is a variant IgG Fc. In someembodiments, the FC polypeptide is a variant canine IgG-A Fc, a variantcanine IgG-B Fc, a variant canine IgG-C Fc, a variant canine IgG-D Fc.In some embodiments, the Fc is a variant equine IgG1 Fc, a variantequine IgG2 Fc, a variant equine IgG3 Fc, a variant equine IgG4 Fc, avariant equine IgG5 Fc, a variant equine IgG6 Fc, or a variant equineIgG7 Fc. In some embodiments, the Fc is a variant feline IgG1a Fc, avariant feline IgG1b Fc, or a variant feline IgG2 Fc.

In some embodiments, L1 and L2, if present, each independently is aflexible linker. In some embodiments, the amino acid sequence of L1 andL2, if present, each independently comprises 100%, at least 95%, atleast 90%, at least 85% serine and/or glycine amino acid residues.

In some embodiments, the contiguous polypeptide comprises an extensionat its C-terminus. In some embodiments, the contiguous polypeptidecomprises a glycine residue, two glycine residues, three glycineresidues, four glycine residues, five glycine residues, six glycineresidues, seven glycine residues, eight glycine residues, or greaterthan eight glycine residues at its C-terminus. In some embodiments, thecontiguous polypeptide comprises an amino acid sequence of SEQ ID NO:88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ IDNO: 93, SEQ ID NO: 94, or SEQ ID NO: 95 at its C-terminus.

In some embodiments, the contiguous polypeptide comprises the amino acidsequence of SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26;SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO:31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ IDNO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 45;SEQ ID NO: 46; SEQ ID NO: 47, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ IDNO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 103; SEQ ID NO: 104;SEQ ID NO: 105; or SEQ ID NO: 106.

A nucleotide sequence encoding a polypeptide of interest, such as avariant IgG Fc polypeptide or other polypeptide described herein, can beinserted into an expression vector suitable for expression in a selectedhost cell. A variant IgG Fc polypeptide or other polypeptide describedherein may be expressed by culturing a host cell transfected with anexpression vector comprising the nucleotide sequence.

A “vector” is a plasmid that can be used to transfer DNA sequences fromone organism to another or to express a gene of interest. A vectortypically includes an origin of replication and regulatory sequenceswhich regulate the expression of the gene of interest, and may or maynot carry a selective marker gene, such as an antibiotic resistancegene. A vector is suitable for the host cell in which it is to beexpressed. A vector may be termed a “recombinant vector” when the geneof interest is present in the vector.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NS0 cells, PER.C6® cells(Crucell), 293 cells, and CHO cells, and their derivatives, such as293-6E, DG44, CHO-S, and CHO-K cells. Host cells include progeny of asingle host cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic DNA complement) to the originalparent cell due to natural, accidental, or deliberate mutation. A hostcell includes cells transfected in vivo with a polynucleotide(s)encoding an amino acid sequence(s) provided herein.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature or produced. For example, a polypeptide isreferred to as “isolated” when it is separated from at least some of thecomponents of the cell in which it was produced. Where a polypeptide issecreted by a cell after expression, physically separating thesupernatant containing the polypeptide from the cell that produced it isconsidered to be “isolating” the polypeptide. Similarly, apolynucleotide is referred to as “isolated” when it is not part of thelarger polynucleotide (such as, for example, genomic DNA ormitochondrial DNA, in the case of a DNA polynucleotide) in which it istypically found in nature, or is separated from at least some of thecomponents of the cell in which it was produced, for example, in thecase of an RNA polynucleotide. Thus, a DNA polynucleotide that iscontained in a vector inside a host cell may be referred to as“isolated.”

A “signal sequence” refers to a sequence of amino acid residues orpolynucleotides encoding such, which facilitates secretion of apolypeptide of interest and is typically cleaved upon export of thepolypeptide to the outside of the cell surface membrane.

In some embodiments, a variant IgG Fc polypeptide or a contiguouspolypeptide comprising a variant Fc polypeptide is isolated usingchromatography, such as size exclusion chromatography, ion exchangechromatography, protein A column chromatography, hydrophobic interactionchromatography, and CHT chromatography.

A label can be attached to a variant IgG Fc polypeptides or a contiguouspolypeptide comprising a variant Fc polypeptide. A “label” means amoiety attached to a molecule to render it detectable. In someembodiments, a variant IgG Fc polypeptide or a contiguous polypeptidecomprising a variant Fc polypeptide is labeled with a detectable moietyincluding but not limited to radioisotopes, fluorescent labels, andvarious enzyme-substrate labels known in the art. In some embodiments,the label is a detectable marker that can produce a signal that isdetectable by visual or instrumental means, for example, incorporationof a radiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (for example,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (for example, ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); chromogens, fluorescentlabels (for example, FITC, rhodamine, lanthanide phosphors), enzymaticlabels (for example, p-galactosidase, horseradish peroxidase,luciferase, alkaline phosphatase); chemiluminescent markers; biotinylgroups; predetermined polypeptide epitopes recognized by a secondaryreporter (for example, leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags); and magneticagents, such as gadolinium chelates. Representative examples of labelscommonly employed for immunoassays include moieties that produce light,for example, acridinium compounds, and moieties that producefluorescence, for example, fluorescein. In this regard, the moietyitself may not be detectably labeled but may become detectable uponreaction with yet another moiety. General techniques to be used inperforming the various immunoassays noted above are known to those ofordinary skill in the art.

Exemplary Variant IgG Fc Polypeptide Affinity to Protein A and/or C1qand/or CD16

The variant IgG Fc polypeptides described herein may have alteredbinding affinity to Protein A and/or C1q and/or CD16. In someembodiments, a variant IgG Fc polypeptide has increased binding affinityto Protein A relative to the wild-type IgG Fc polypeptide. Such variantIgG Fc polypeptides may be purified by Protein A column chromatography.In some embodiments, a variant IgG Fc polypeptide has reduced bindingaffinity to C1q relative to the wild-type IgG Fc polypeptide. Suchvariant IgG Fc polypeptides may have reduced complement-mediated immuneresponses. In some embodiments, a variant IgG Fc polypeptide has reducedbinding affinity to CD16 relative to the wild-type IgG Fc polypeptide.Such variant IgG Fc polypeptides may have reduced ADCC immune responses.In some embodiments, a variant IgG Fc polypeptide has increased bindingaffinity to Protein A relative to the wild-type IgG Fc polypeptideand/or has reduced binding affinity to C1q relative to the wild-type IgGFc polypeptide and/or has reduced binding affinity to CD16 relative tothe wild-type IgG Fc polypeptide.

“Protein A,” as used herein, is a polypeptide comprising the entirety ora portion of Protein A that is capable of binding a wild-type canineIgG-B Fc, a wild-type equine IgG1 Fc, a wild-type equine IgG3 Fc, awild-type equine IgG4 Fc, a wild-type equine IgG7 Fc, a wild-type felineIgG1a Fc, a wild-type feline IgG1b Fc, or a wild-type feline IgG2 Fc.

“C1q” or “C1q complex” is used interchangeably to refer to a proteincomplex involved in the complement system, or a portion thereof, thatcan bind a wild-type canine IgG-B Fc, a wild-type canine IgG-C Fc, awild-type equine IgG1 Fc, a wild-type equine IgG3 Fc, a wild-type equineIgG4 Fc, a wild-type equine IgG7 Fc, a wild-type feline IgG1a Fc, or awild-type feline IgG1b Fc.

“CD16,” as used herein, is a polypeptide comprising the entirety or aportion of CD16 that is capable of binding a wild-type canine IgG-A Fcor a wild-type canine IgG-D Fc. The term “binds” to a substance is aterm that is well understood in the art, and methods to determine suchbinding are also well known in the art. A molecule is said to exhibit“binding” if it reacts, associates with, or has affinity for aparticular cell or substance and the reaction, association, or affinityis detectable by one or more methods known in the art, such as, forexample, immunoblot, ELISA, KinEx A, biolayer interferometry (BLI),surface plasmon resonance devices, or etc.

“Protein A +,” as used herein, means that the Fc polypeptide has ProteinA binding affinity. In some embodiments, a Protein A+Fc polypeptidecomprises at least one an amino acid modification that increases ProteinA binding affinity.

“Protein A −,” as used herein, means that the Fc polypeptide has low orno Protein A binding affinity.

“C1q+,” as used herein, means that the Fc polypeptide has C1q bindingaffinity.

“C1q −,” as used herein, means that the Fc polypeptide has low or no C1qbinding affinity. In some embodiments, a C1q−Fc polypeptide has at leastone an amino acid modification that reduces C1q binding affinity.

“CD16+,” as used herein, means that the Fc polypeptide has CD16 bindingaffinity.

“CD16−,” as used herein, means that the Fc polypeptide has low or noCD16 binding affinity. In some embodiments, a CD16− Fc polypeptide hasat least one an amino acid modification that reduces CD16 bindingaffinity.

The term “affinity” means the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (for example, areceptor) and its binding partner (for example, a ligand). The affinityof a molecule X for its partner Y can generally be represented by thedissociation constant (Ku). Affinity can be measured by common methodsknown in the art, such as, for example, immunoblot, ELISA, KinEx A,biolayer interferometry (BLI), or surface plasmon resonance devices.

“Surface plasmon resonance” denotes an optical phenomenon that allowsfor the analysis of real-time biospecific interactions by detection ofalterations in protein concentrations within a biosensor matrix, forexample using the BIAcore™ system (BIAcore International AB, a GEHealthcare company, Uppsala, Sweden and Piscataway, N.J.). For furtherdescriptions, see Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26.

“Biolayer interferometry” refers to an optical analytical technique thatanalyzes the interference pattern of light reflected from a layer ofimmobilized protein on a biosensor tip and an internal reference layer.Changes in the number of molecules bound to the biosensor tip causeshifts in the interference pattern that can be measured in real-time. Anonlimiting exemplary device for biolayer interferometry is an Octet®system (Pall ForteBio LLC). See, e.g., Abdiche et al., 2008, Anal.Biochem. 377: 209-277.

The terms “K_(D),” “K_(d),” “Kd” or “Kd value” as used interchangeablyto refer to the equilibrium dissociation constant of a receptor-ligandinteraction or antibody-antigen interaction.

In some embodiments, a variant IgG Fc polypeptide binds to Protein Awith a dissociation constant (K_(D)) of less than 5×10⁻⁶ M, less than1×10⁻⁶ M, less than 5×10⁻⁷ M, less than 1×10⁻⁷ M, less than 5×10⁻⁸ M,less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M, less than5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M, less than 5×10⁻¹¹ M, less than 1×10⁻¹¹M, less than 5×10⁻¹² M, or less than 1×10⁻¹² M, as measured by biolayerinterferometry.

In some embodiments, a variant IgG Fc polypeptide binds to C1q or CD16with a dissociation constant (K_(D)) of greater than 5×10⁻⁶ M, greaterthan 1×10⁻⁵ M, greater than 5×10⁻⁵ M, greater than 1×10⁻⁴ M, greaterthan 5×10⁻⁴ M, or greater than 1×10⁻³ M, as measured by biolayerinterferometry.

In some embodiments, the K_(D) of an IgG Fc polypeptide, such as avariant IgG Fc polypeptide, to Protein A or to C1q or to CD16 ismeasured by using biolayer interferometry assays using a biosensor, suchas an Octet® System (Pall ForteBio LLC, Fremont, Calif.) according tothe supplier's instructions. In brief, biotinylated Protein A or C1q orCD16 is bound to the sensor tip and the association of IgG Fcpolypeptide is monitored for a specified time or until steady state isreached. Dissociation may be monitored for a specified time or untilsteady state is reached. A buffer only blank curve is subtracted tocorrect for any drift. The data are fit to a 2:1 binding model usingForteBio data analysis software to determine association rate constant(k_(on)), dissociation rate constant (k_(off)), and the K_(d). Theequilibrium dissociation constant (K_(D)) is calculated as the ratio ofk_(off)/k_(on). The term “k_(on)” refers to the rate constant forassociation of a molecule X to its partner Y and the term “k_(off)”refers to the rate constant for dissociation of a molecule X or partnerY from the molecule X/partner Y complex.

To “increase” or “stimulate” means to increase, improve, or augment anactivity, function, or amount as compared to a reference. In someembodiments, by “increase” or “stimulate” is meant the ability to causean overall increase of about 5% or greater, of about 10% or greater, ofabout 20% or greater, of about 30% or greater, of about 40% or greater,of about 50% or greater, of about 60% or greater, of about 70% orgreater, of about 80% or greater, of about 90% or greater, of about 100%or greater, of about 125% or greater, of about 200% or greater relativeto a reference value. In some embodiments, by “increase” or “stimulate”is meant the ability to cause an overall increase of about 5% to about50%, of about 10% to about 20%, of about 50% to about 100%, of about 25%to about 70% relative to a reference value. In some embodiments, by“increase” or “stimulate” is meant the ability to cause an overallincrease of 50% or greater. In some embodiments, by “increase” or“stimulate” is meant the ability to cause an overall increase of 75%,85%, 90%, 95%, or greater. In some embodiments, the amount noted aboveis stimulated or increased over a period of time, relative to a controldose (such as a placebo) over the same period of time.

In some embodiments, a variant IgG Fc polypeptide is capable of bindingto Protein A with an increased affinity of about 5% or greater, of about10% or greater, of about 20% or greater, of about 30% or greater, ofabout 40% or greater, of about 50% or greater, of about 60% or greater,of about 70% or greater, of about 80% or greater, of about 90% orgreater, of about 100% or greater, of about 125% or greater, of about150% or greater, of about 200% or greater relative to a reference IgG Fcpolypeptide. In some embodiments, a variant IgG Fc polypeptide iscapable of binding to Protein A with an increased affinity of about 5%to about 50%, of about 10% to about 20%, of about 50% to about 100%, ofabout 25% to about 70% relative to a reference IgG Fc polypeptide. Insome embodiments, the reference IgG Fc polypeptide is a wild-type IgG Fcpolypeptide. In some embodiments, the reference IgG Fc polypeptide is adifferent variant IgG Fc polypeptide.

To “reduce” or “inhibit” means to decrease, reduce, or arrest anactivity, function, or amount as compared to a reference. In someembodiments, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of about 5% or greater, of about 10% or greater, ofabout 20% or greater, of about 30% or greater, of about 40% or greater,of about 50% or greater, of about 60% or greater, of about 70% orgreater, of about 80% or greater, or of about 90% or greater relative toa reference IgG Fc polypeptide. In some embodiments, by “reduce” or“inhibit” is meant the ability to cause an overall decrease of about 5%to about 50%, of about 10% to about 20%, of about 50% to about 100%, ofabout 25% to about 70% relative to a reference value. In someembodiments, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of 50% or greater. In some embodiments, by “reduce” or“inhibit” is meant the ability to cause an overall decrease of 75%, 85%,90%, 95%, or greater. In some embodiments, the amount noted above isinhibited or decreased over a period of time, relative to a control dose(such as a placebo) over the same period of time.

In some embodiments, a variant IgG Fc polypeptide is capable of bindingto C1q or CD16 with a decreased affinity of about 5% or greater, ofabout 10% or greater, of about 20% or greater, of about 30% or greater,of about 40% or greater, of about 50% or greater, of about 60% orgreater, of about 70% or greater, of about 80% or greater, of about 90%or greater relative to a reference IgG Fc polypeptide. In someembodiments, a variant IgG Fc polypeptide is capable of binding to C1qor CD16 with a decreased affinity of about 5% to about 50%, of about 10%to about 20%, of about 50% to about 100%, of about 25% to about 70%relative to a reference IgG Fc polypeptide. In some embodiments, thereference IgG Fc polypeptide is a wild-type IgG Fc polypeptide. In someembodiments, the reference IgG Fc polypeptide is a different variant IgGFc polypeptide.

A “reference” as used herein, refers to any sample, standard, or levelthat is used for comparison purposes. A reference may be a wild-typereference or a variant reference. A reference may be obtained from ahealthy or non-diseased sample. In some examples, a reference isobtained from a non-diseased or non-treated sample of a companionanimal. In some examples, a reference is obtained from one or morehealthy animals of a particular species, which are not the animal beingtested or treated.

Exemplary Biological Activity of Variant GLP1 Fusion Molecules

In some embodiments, a GLP1 fusion molecule binds to GLP1R and activatescAMP production. In some embodiments, a GLP1 fusion polypeptideincreases production of cAMP in a cell by at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or 100% compared to signaling function in the absence of theGLP1 fusion polypeptide.

Exemplary Pharmaceutical Compositions

The terms “pharmaceutical formulation” and “pharmaceutical composition”refer to a preparation which is in such form as to permit the biologicalactivity of the active ingredient(s) to be effective, and which containsno additional components that are unacceptably toxic to a subject towhich the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed. Examples of pharmaceutically acceptable carriers includealumina; aluminum stearate; lecithin; serum proteins, such as humanserum albumin, canine or other animal albumin; buffers such asphosphate, citrate, tromethamine or HEPES buffers; glycine; sorbic acid;potassium sorbate; partial glyceride mixtures of saturated vegetablefatty acids; water; salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, or magnesium trisilicate;polyvinyl pyrrolidone, cellulose-based substances; polyethylene glycol;sucrose; mannitol; or amino acids including, but not limited to,arginine.

The pharmaceutical composition can be stored in lyophilized form. Thus,in some embodiments, the preparation process includes a lyophilizationstep. The lyophilized composition may then be reformulated, typically asan aqueous composition suitable for parenteral administration, prior toadministration to the dog, cat, or horse. In other embodiments,particularly where a variant IgG Fc polypeptide or other polypeptidedescribed herein is highly stable to thermal and oxidative denaturation,the pharmaceutical composition can be stored as a liquid, i.e., as anaqueous composition, which may be administered directly, or withappropriate dilution, to the dog, cat, or horse. A lyophilizedcomposition can be reconstituted with sterile Water for Injection (WFI).Bacteriostatic reagents, such benzyl alcohol, may be included. Thus, theinvention provides pharmaceutical compositions in solid or liquid form.

The pH of the pharmaceutical compositions may be in the range of fromabout pH 5 to about pH 8, when administered. The compositions of theinvention are sterile if they are to be used for therapeutic purposes.Sterility can be achieved by any of several means known in the art,including by filtration through sterile filtration membranes (e.g., 0.2micron membranes). Sterility may be maintained with or withoutanti-bacterial agents.

Certain Uses of Fc Polypeptides and Pharmaceutical Compositions

A polypeptide comprising a variant Fc polypeptide, such as a variant IgGFc polypeptide, of the invention or pharmaceutical compositionscomprising a variant Fc polypeptide of the invention may be useful forextending product half-life in vivo in a companion animal, including,but not limited to, canine, feline, or equine.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. “Treatment” as used herein, covers anyadministration or application of a therapeutic for disease in a mammal,including a companion animal. For purposes of this disclosure,beneficial or desired clinical results include, but are not limited to,any one or more of: alleviation of one or more symptoms, diminishment ofextent of disease, preventing or delaying spread of disease, preventingor delaying recurrence of disease, delay or slowing of diseaseprogression, amelioration of the disease state, inhibiting the diseaseor progression of the disease, inhibiting or slowing the disease or itsprogression, arresting its development, and remission (whether partialor total). Also encompassed by “treatment” is a reduction ofpathological consequence of a proliferative disease. The methodsprovided herein contemplate any one or more of these aspects oftreatment. In-line with the above, the term treatment does not requireone-hundred percent removal of all aspects of the disorder.

A “therapeutically effective amount” of a substance/molecule, agonist orantagonist may vary according to factors such as the type of disease tobe treated, the disease state, the severity and course of the disease,the type of therapeutic purpose, any previous therapy, the clinicalhistory, the response to prior treatment, the discretion of theattending veterinarian, age, sex, and weight of the animal, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the animal. A therapeutically effective amount isalso one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A therapeutically effective amountmay be delivered in one or more administrations. A therapeuticallyeffective amount refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

In some embodiments, a variant IgG Fc polypeptide or other polypeptidedescribed herein, or a pharmaceutical composition comprising such isadministered parenterally, by subcutaneous administration, intravenousinfusion, or intramuscular injection. In some embodiments, a variant IgGFc polypeptide or other polypeptide described herein, or apharmaceutical composition comprising such is administered as a bolusinjection or by continuous infusion over a period of time. In someembodiments, a variant IgG Fc polypeptide or other polypeptide describedherein, or a pharmaceutical composition comprising such is administeredby an intramuscular, an intraperitoneal, an intracerebrospinal, asubcutaneous, an intra-arterial, an intrasynovial, an intrathecal, or aninhalation route.

In some embodiments, a GLP1 fusion polypeptide or pharmaceuticalcompositions comprising it can be utilized in accordance with themethods herein to treat high blood glucose-related conditions. In someembodiments, an GLP1 fusion polypeptide or pharmaceutical compositionsis administered to a companion animal, such as a canine, a feline, orequine, to treat high blood glucose-related condition.

In some embodiments, a variant IgG Fc polypeptide or other polypeptidedescribed herein, or a pharmaceutical composition comprising such isadministered in an amount in the range of 0.0001 mg/kg body weight to100 mg/kg body weight per dose. In some embodiments, GLP1 analog may beadministered in an amount in the range of 0.005 mg/kg body weight to 20mg/kg body weight per dose. In some embodiments, GLP1 analog may beadministered in an amount in the range of 1 mg/kg body weight to 10mg/kg body weight per dose. In some embodiments, GLP1 analog may beadministered in an amount in the range of 0.5 mg/kg body weight to 100mg/kg body, in the range of 1 mg/kg body weight to 100 mg/kg bodyweight, in the range of 5 mg/kg body weight to 100 mg/kg body weight, inthe range of 10 mg/kg body weight to 100 mg/kg body weight, in the rangeof 20 mg/kg body weight to 100 mg/kg body weight, in the range of 50mg/kg body weight to 100 mg/kg body weight, in the range of 1 mg/kg bodyweight to 10 mg/kg body weight, in the range of 5 mg/kg body weight to10 mg/kg body weight, in the range of 0.5 mg/kg body weight to 10 mg/kgbody weight, or in the range of 5 mg/kg body weight to 50 mg/kg bodyweight.

In some embodiments, a variant IgG Fc polypeptide or other polypeptidedescribed herein, or a pharmaceutical composition comprising such isadministered to a companion animal at one time or over a series oftreatments. In some embodiments, the dose is administered once per weekfor at least two or three consecutive weeks, and in some embodiments,this cycle of treatment is repeated two or more times, optionallyinterspersed with one or more weeks of no treatment. In otherembodiments, the therapeutically effective dose is administered once perday for two to five consecutive days, and in some embodiments, thiscycle of treatment is repeated two or more times, optionallyinterspersed with one or more days or weeks of no treatment.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive or sequentialadministration in any order. The term “concurrently” is used herein torefer to administration of two or more therapeutic agents, where atleast part of the administration overlaps in time or where theadministration of one therapeutic agent falls within a short period oftime relative to administration of the other therapeutic agent. Forexample, the two or more therapeutic agents are administered with a timeseparation of no more than about a specified number of minutes. The term“sequentially” is used herein to refer to administration of two or moretherapeutic agents where the administration of one or more agent(s)continues after discontinuing the administration of one or more otheragent(s), or wherein administration of one or more agent(s) beginsbefore the administration of one or more other agent(s). For example,administration of the two or more therapeutic agents are administeredwith a time separation of more than about a specified number of minutes.As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the animal.

In some embodiments, the dose is administered once per week for at leasttwo or three consecutive weeks, and in some embodiments, this cycle oftreatment is repeated two or more times, optionally interspersed withone or more weeks of no treatment. In other embodiments, thetherapeutically effective dose is administered once per day for two tofive consecutive days, and in some embodiments, this cycle of treatmentis repeated two or more times, optionally interspersed with one or moredays or weeks of no treatment.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive or sequentialadministration in any order. The term “concurrently” is used herein torefer to administration of two or more therapeutic agents, where atleast part of the administration overlaps in time or where theadministration of one therapeutic agent falls within a short period oftime relative to administration of the other therapeutic agent. Forexample, the two or more therapeutic agents are administered with a timeseparation of no more than about a specified number of minutes. The term“sequentially” is used herein to refer to administration of two or moretherapeutic agents where the administration of one or more agent(s)continues after discontinuing the administration of one or more otheragent(s), or wherein administration of one or more agent(s) beginsbefore the administration of one or more other agent(s). For example,administration of the two or more therapeutic agents are administeredwith a time separation of more than about a specified number of minutes.As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the animal.

In some embodiments, the method comprises administering in combinationwith a GLP1 fusion polypeptide insulin, a DPP4 inhibitor, a SGLT2inhibitor, a biguanides sulfonylurea, a meglitinide derivative, analpha-glucosidase inhibitor, a thiazolidinedione (TZD), anamylinomimetic, a bile acid sequestrant, a dopamine agonist.

The following examples illustrate particular aspects of the disclosureand are not intended in any way to limit the disclosure.

EXAMPLES Example 1 Variant Canine IgG Fc Polypeptides for IncreasedProtein a Binding and/or Decreased Complement Binding and/or DecreasedCD16 Binding

Purification of antibodies using Protein A affinity is a well-developedprocess. However, among four subtypes of canine IgG, only IgG-B Fc(e.g., SEQ ID NO: 2 or SEQ ID NO: 107) has Protein A binding affinity.Canine IgG-A Fc (e.g., SEQ ID NO: 1), IgG-C Fc (e.g., SEQ ID NO: 3 orSEQ ID NO: 108), and IgG-D Fc (e.g., SEQ ID NO: 4) have weak or nomeasurable Protein A binding affinity. Variant canine IgG-A Fc, IgG-CFc, and IgG-D Fc polypeptides were designed for altered Protein Abinding.

In addition, canine IgG-B Fc and IgG-C Fc have complement activity andbind to C1q, while canine IgG-A Fc and IgG-D Fc have weak or nomeasurable binding affinity to C1q. To potentially reduce the C1qbinding and/or potentially reduce complement-mediated immune responses,variant canine IgG-B Fc and IgG-C Fc polypeptides were designed.

Furthermore, canine IgG-B Fc and IgG-C Fc have CD16 binding activity. Topotentially reduce the binding of CD16 to IgG-B Fc and IgG-C Fc, and/orpotentially reduce ADCC, variant canine IgG-B Fc and IgG-C Fcpolypeptides were designed.

Table 3, below summarizes the Protein A and C1q binding characteristicsof canine IgG Fc subtypes. Notably, none of the wild-type canine IgG Fcsubtypes lacks C1q binding and binds Protein A.

TABLE 3 Wild-type Protein A C1q CD16 Canine IgG Fc Binding BindingBinding IgG-A Fc − − − IgG-B Fc + + + IgG-C Fc − + + IgG-D Fc − − − (−)denotes low or no measurable binding activity.

Using three-dimensional protein modeling and protein sequence analysis,the sequences of canine IgG-B Fc that are likely in contact with ProteinA were identified. FIG. 1 shows an alignment of canine IgG-A, B, C, andD Fc sequences. The boxes indicate the regions likely in contact withProtein A.

Two approaches were used to design variant canine IgG-A, IgG-C, andIgG-D Fc polypeptides for increased Protein A binding. For the firstapproach, variant canine IgG-A, IgG-C, and IgG-D Fc polypeptides weredesigned to have the same Protein A binding motif sequences as canineIgG-B Fc (e.g., SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7,respectively). For the second approach, variant canine IgG-A FcI(21)T/Q(207)H (SEQ ID NO: 60), variant canine IgG-C Fc I(21)T (SEQ IDNO: 61), and variant canine IgG-D Fc I(21)T/Q(207)H (SEQ ID NO: 62) weredesigned with one or two amino acid substitutions in the Protein Abinding region to correspond with the canine IgG-B Fc sequence.

In addition, variant canine IgG-A Fc, IgG-C Fc, and IgG-D Fcpolypeptides with increased Protein A binding may be prepared having oneor more of the amino acid substitutions listed in Table 4.

TABLE 4 Variant Canine IgG Fc Amino Acid Substitutions* (Protein A +)Canine IgG-A Fc Canine IgG-C Fc Canine IgG-D Fc (SEQ ID NO: 1) (SEQ IDNO: 3) (SEQ ID NO: 4) Ile (21) Thr Ile (21) Thr Ile (23) Thr Arg (23)Leu Val (23) Leu Arg (23) Leu Thr (25) Ala Thr (24) Ile Thr (25) Ala Glu(80) Gly Glu (80) Gly Thr (205) Ala Gln (207) His Gln (207) His *Theamino acid positions listed are relative to the SEQ ID NO. indicated.

To potentially reduce the binding of C1q to canine IgG-B Fc and IgG-CFc, and/or potentially reduce complement-mediated immune responses,variant canine IgG-B Fc and IgG-C Fc polypeptides may be prepared havingan amino acid substitution of Lys with any amino acid except Lys at anamino acid position corresponding to position 93 of SEQ ID NO: 2 or ofSEQ ID NO: 3, respectively. These amino acid substitutions wereidentified after analysis of the protein sequence and 3-D structuremodeling of canine IgG-B Fc and IgG-C Fc compared to canine IgG-A Fc andIgG-D Fc, which are understood to not exhibit complement activity. Forexample, variant canine IgG-B Fc K(93)R (SEQ ID NO: 78) and variantcanine IgG-C Fc K(93)R (SEQ ID NO: 79) may be prepared. Reduced bindingbetween human C1q and a fusion protein comprising variant canine IgG-BFc K(93)R was observed when compared to a fusion protein comprisingwild-type canine IgG-B Fc.

To potentially reduce the binding of CD16 to IgG-B Fc and IgG-C Fc,and/or potentially reduce ADCC, variant canine IgG-B Fc and IgG-C Fcpolypeptides may be prepared having one or more of the amino acidsubstitutions listed in Table 5. The amino acid substitution(s) wereidentified after analysis of the protein sequence and 3-D structuremodeling of canine IgG-B and IgG-C compared to IgG-A and IgG-D, whichare understood to not exhibit ADCC activity.

TABLE 5 Original residue position* Canine IgG-B Fc Canine IgG-C Fc (SEQID NO: 2) (SEQ ID NO: 3) Substitution(s) Met (5) Leu (5) Any amino acidexcept original residue, such as Pro Asp (38) Asp (38) Any amino acidexcept original residue, such as Gly Pro (39) Pro (39) Any amino acidexcept original residue, such as Arg Lys (97) Lys (97) Any amino acidexcept original residue, such as Ile Ala (98) Ala (98) Any amino acidexcept original residue, such as Gly *The amino acid positions listedare relative to the SEQ ID NO. indicated.

Since wild-type canine IgG-C Fc lacks Protein A binding and has C1qbinding, a double variant canine IgG-C Fc that binds Protein A and hasreduced binding to C1q may be prepared by combining one or more of theamino acid substitutions listed in Table 4 with a K(93)R substitution orK(93)X substitution, wherein X is any amino acid except Lys. A doublevariant canine IgG-B Fc or double variant canine IgG-C Fc with reducedbinding to C1q and reduced binding to CD16 may be prepared by combiningone or more of the amino acid substitutions listed in Table 5 with aK(93)R substitution or K(93)X substitution, wherein X is any amino acidexcept Lys. A triple variant canine-IgG-C Fc that binds Protein A andhas reduced binding to C1q and CD16 may be prepared by combining one ormore of the amino acid substitutions listed in Table 4 and one or moreof the amino acid substitutions listed in Table 5 with a K(93)Rsubstitution or K(93)X substitution, wherein X is any amino acid exceptLys.

The binding of any variant canine IgG Fc to Protein A, CD16, and/or C1qmay be determined and compared to the binding of another IgG Fc toProtein A, CD16, and/or C1q (e.g., the corresponding wild-type canineIgG Fc, another wild-type or variant canine IgG Fc, or a wild-type orvariant IgG Fc of another companion animal, etc.).

Binding analysis may be performed using an Octet biosensor. Briefly, thetarget molecule (e.g., Protein A, C1q, CD16, etc.) may be biotinylatedand free unreacted biotin removed (e.g., by dialysis). The biotinylatedtarget molecule is captured on streptavidin sensor tips. Association ofthe target molecule with various concentrations (e.g., 10 μg/mL) of IgGFc polypeptide is monitored for a specified time or until steady stateis reached. Dissociation is monitored for a specified time or untilsteady state is reached. A buffer only blank curve may be subtracted tocorrect for any drift. The data are fit to a 1:1 binding model usingForteBio™ data analysis software to determine the k_(on), k_(off), andthe K_(d).

Example 2 Variant Equine IgG Fc Polypeptides for Increased Protein aBinding and/or Decreased Complement Binding

Of the seven subtypes of equine IgG, IgG1 Fc (e.g., SEQ ID NO: 63), IgG3Fc (e.g., SEQ ID NO: 65), IgG4 Fc (e.g., SEQ ID NO: 66), IgG7 Fc (e.g.,SEQ ID NO: 69) have Protein A binding affinity. Equine IgG2 Fc (e.g.,SEQ ID NO: 18, SEQ ID NO: 64), IgG5 Fc (e.g., SEQ ID NO: 67), and IgG6Fc (e.g., SEQ ID NO: 68) have weak or no measurable Protein A bindingaffinity. Variant equine IgG2 Fc, IgG5 Fc, and IgG6 Fc polypeptides weredesigned for altered Protein A binding.

In addition, equine IgG2 Fc, IgG5 Fc, and IgG6 Fc have weak or nomeasurable binding affinity to C1q, while equine IgG1 Fc, IgG3 Fc, IgG4Fc, and IgG7 Fc bind to C1q. To potentially reduce the C1q bindingand/or potentially reduce complement-mediated immune responses, variantequine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc polypeptides weredesigned.

Table 6, below summarizes the Protein A and C1q binding characteristicsof equine IgG Fc subtypes. Notably, none of the wild-type equine IgG Fcsubtypes lacks C1q binding and binds Protein A.

TABLE 6 Wild-type Protein A C1q Equine IgG Fc Binding Binding IgG1Fc + + IgG2 Fc − − IgG3 Fc + + IgG4 Fc + + IgG5 Fc − − IgG6 Fc − − IgG7Fc + + (−) denotes low or no measurable binding activity.

Using three-dimensional protein modeling and protein sequence analysis,the sequences of equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc that arelikely in contact with Protein A were identified. Variant equine IgG2Fc, IgG5 Fc, and IgG6 Fc polypeptides with increased Protein A bindingmay be prepared having one or more of the amino acid substitutionslisted in Table 7.

TABLE 7 Variant Equine IgG Fc Amino Acid Substitutions* (Protein A +)Equine IgG2 Fc Equine IgG5 Fc Equine Ig6 Fc (SEQ ID NO: 64) (SEQ ID NO:67) (SEQ ID NO: 68) Ala (15) Thr Val (199) Leu Ile (199) Leu Phe (203)Tyr Glu (200) Tyr Arg (200) His His (201) Asn Thr (202) His *The aminoacid positions listed are relative to the SEQ ID NO. indicated

For example, variant equine IgG2 Fc, IgG5 Fc, and IgG6 Fc polypeptideswere designed with one or multiple amino acid substitutions in theProtein A binding region to correspond with the sequence of wild-typeequine IgG Fc, which does bind Protein A. Variant equine IgG2 Fc F(203)Y(SEQ ID NO: 71); variant equine IgG2 Fc A(15)T/F(203)Y (SEQ ID NO: 72);variant equine IgG5 Fc V(199)L/E(200)Y (SEQ ID NO: 75); and variantequine IgG6 Fc I(199)L/R(200)H/H(201)N/T(202)H (SEQ ID NO: 76) withincreased Protein A binding may be prepared.

To potentially reduce the binding of C1q to equine IgG1 Fc, IgG3 Fc,IgG4 Fc, and IgG7 Fc, and/or potentially reduce complement-mediatedimmune responses, variant canine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fcpolypeptides may be prepared having an amino acid substitution of Lyswith any amino acid except Lys at an amino acid position correspondingto position 87 of SEQ ID NO: 63, of SEQ ID NO: 65, of SEQ ID NO: 66, ofSEQ ID NO: 69, respectively. These amino acid substitutions wereidentified after analysis of the protein sequence and 3-D structuremodeling of equine IgG1 Fc, IgG3 Fc, IgG4 Fc, and IgG7 Fc compared toequine IgG2 Fc, IgG5 Fc, and IgG6 Fc, which are understood to notexhibit complement activity. For example, variant equine IgG1 Fc K(87)S(SEQ ID NO: 70), variant equine IgG 3 Fc K(87)S (SEQ ID NO: 73), variantequine IgG4 Fc K(87)S (SEQ ID NO: 74), and variant equine IgG7 Fc K(87)S(SEQ ID NO: 77) may be prepared.

The binding of any variant equine IgG Fc to Protein A and/or C1q may bedetermined and compared to the binding of another IgG Fc to Protein Aand/or C1q (e.g., the corresponding wild-type equine IgG Fc, anotherwild-type or variant equine IgG Fc, or a wild-type or variant IgG Fc ofanother companion animal, etc.). The binding assay described in Example1 may be used.

Example 3 Variant Feline IgG Fc Polypeptides for Decreased ComplementBinding

Each of the three subtypes of feline IgG, IgG1a Fc (SEQ ID NO: 80 or SEQID NO: 117), IgG1b Fc (SEQ ID NO: 81 or SEQ ID NO: 118), and IgG2 Fc(SEQ ID NO: 16) have Protein A binding affinity. However, only felineIgG2 Fc has weak or no measurable binding affinity to C1q, while felineIgG1a Fc, IgG1b Fc bind to C1q. To potentially reduce the C1q bindingand/or potentially reduce complement-mediated immune responses, variantfeline IgG1a Fc and IgG1b Fc polypeptides were designed.

Table 8, below summarizes the Protein A and C1q binding characteristicsof feline IgG Fc subtypes. Notably, none of the wild-type equine IgG Fcsubtypes lacks C1q binding and binds Protein A.

TABLE 8 Wild-type Protein A C1q Feline IgG Fc Binding Binding IgG1aFc + + IgG1b Fc + + IgG2 Fc + − (−) denotes low or no measurable bindingactivity.

To potentially reduce the binding of C1q to feline IgG1a Fc and IgG1bFc, and/or potentially reduce complement-mediated immune responses,variant feline IgG1a Fc and IgG1b Fc polypeptides may be prepared havingan amino acid substitution of Pro with any amino acid except Pro at anamino acid position corresponding to position 198 of SEQ ID NO: 80 or ofSEQ ID NO: 81, respectively. These amino acid substitutions wereidentified after analysis of the protein sequence and 3-D structuremodeling of feline IgG1a Fc and IgG1b Fc compared to feline IgG2 Fc,which is understood to not exhibit complement activity. For example,variant feline IgG1a Fc P(198)A (SEQ ID NO: 82) and variant feline IgG1bFc P(198)A (SEQ ID NO: 83) may be prepared.

The binding of any variant feline IgG Fc to C1q may be determined andcompared to the binding of another IgG Fc to C1q (e.g., thecorresponding wild-type feline IgG Fc, another wild-type or variantfeline IgG Fc, or a wild-type or variant IgG Fc of another companionanimal, etc.). The binding assay described in Example 1 may be used.

Example 4 Variant Canine and Feline IgG Fc Polypeptides forHeterodimeric Proteins

To enable the preparation of a bispecific canine or feline antibody or abifunctional canine or feline Fc fusion protein using a knob-in-holeheterodimerization approach, pairing of variant canine IgG Fcpolypeptides and variant feline IgG Fc polypeptides was investigated.

An amino acid substitution of threonine at a position corresponding toposition 138 of canine IgG-A (SEQ ID NO: 1), at a position correspondingto position 137 of canine IgG-B Fc (SEQ ID NO: 2), at a positioncorresponding to position 137 of canine IgG-C Fc (SEQ ID NO: 3), or at aposition corresponding to position 138 of canine IgG-D Fc (SEQ ID NO: 4)to tyrosine (T138Y or T137Y) can be introduced to one Fc chain(heterodimer chain 1). Examples of amino acid sequences of variantcanine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 1are SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14,respectively.

An amino acid substitution of tyrosine at a position corresponding toposition 181 of canine IgG-A (SEQ ID NO: 1), at a position correspondingto position 180 of canine IgG-B Fc (SEQ ID NO: 2), at a positioncorresponding to position 180 of canine IgG-C Fc (SEQ ID NO: 3), or at aposition corresponding to position 181 of canine IgG-D Fc (SEQ ID NO: 4)to threonine (Y181T or Y180T) can be introduced to a second Fc chain(heterodimer chain 2). Examples of amino acid sequences of variantcanine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 2are SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15,respectively.

A second pairing of variant canine IgG Fc heterodimer chains 3 and 4 wasalso investigated. An amino acid substitution of threonine to tryptophanat a position corresponding to position 138 of canine IgG-A (SEQ IDNO: 1) or of canine IgG-D (SEQ ID NO: 4) (T138W), or at a positioncorresponding to position 137 of canine IgG-B Fc (SEQ ID NO: 2) or ofcanine IgG-C Fc (SEQ ID NO: 3) (T137W) can be introduced to one Fc chain(heterodimer chain 3). Examples of amino acid sequences of variantcanine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fc heterodimer chain 3are SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, and SEQ ID NO: 115.

An amino acid substitution of threonine to serine at a positioncorresponding to position 138, of leucine to alanine at a positioncorresponding to position 140, and of tyrosine to threonine at aposition corresponding to position 181 of canine IgG-A (SEQ ID NO: 1) orof IgG-D (SEQ ID NO: 4) (T138S, L140A, Y181T), or of threonine to serineat a position corresponding to position 137, of leucine to alanine at aposition corresponding to position 139, and of tyrosine to threonine ata position corresponding to position 180 of canine IgG-B Fc (SEQ ID NO:2) or of IgG-C(SEQ ID NO: 3) (T137S, L139A, Y180T) can be introduced toa second Fc chain (heterodimer chain 4). Examples of amino acidsequences of variant canine IgG-A Fc, IgG-B Fc, IgG-C Fc, and IgG-D Fcheterodimer chain 4 are SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114,and SEQ ID NO: 116.

An amino acid substitution of threonine to tryptophan at a positioncorresponding to position 154 of feline IgG2 (SEQ ID NO: 16), of felineIgG1a Fc (SEQ ID NO: 80 or SEQ ID NO: 117), or of feline IgG1b Fc (SEQID NO: 81 or SEQ ID NO: 118) (T154W) can be introduced to one Fc chain(heterodimer chain 1). Examples of amino acid sequences of variantfeline IgG2 Fc, IgG1a Fc, and IgG1b Fc heterodimer chain 1 are SEQ IDNO: 119, SEQ ID NO: 121, and SEQ ID NO: 123, respectively.

An amino acid substitution of threonine to serine at a positioncorresponding to position 154, of leucine to alanine at a positioncorresponding to position 156, and of tyrosine to threonine at aposition corresponding to position 197 of feline IgG2 Fc (SEQ ID NO:16), of IgG-1a (SEQ ID NO: 80 or SEQ ID NO: 117), or of IgG-1b Fc (SEQID NO: 81 or SEQ ID NO: 118) (T154S, L156A, Y197T) can be introduced toa second Fc chain (heterodimer chain 4). Examples of amino acidsequences of variant feline IgG2 Fc, IgG1a Fc, and IgG1b Fc heterodimerchain 4 are SEQ ID NO: 120, SEQ ID NO: 122, and SEQ ID NO: 124.

The pairing of variant canine IgG Fc heterodimer chains 1 and 2, thepairing of variant canine IgG Fc heterodimer chains 3 and 4, or thepairing of variant feline IgG Fc heterodimer chains 1 and 2 may allowfor Fc heterodimerization and prevent or reduce Fc homodimerization. Aheterodimer chain 1 of one canine IgG subtype may be combined with aheterodimer chain 2 of the same or a different canine IgG subtype. Aheterodimer chain 3 of one canine IgG subtype may be combined with aheterodimer chain 4 of the same or a different canine IgG subtype. Aheterodimer chain 1 of one feline IgG subtype may be combined with aheterodimer chain 2 of the same or a different feline IgG subtype. Thedesign can enable dimerization of bispecific canine or felineantibodies. In addition, two different peptides or proteins or acombination of different proteins can be fused to the heterodimeric Fcchains. For example, a dual GLP1 and glucagon molecule can be createdusing variant canine IgG Fc heterodimer chains or variant feline IgG Fcheterodimer chains, such as a GLP1 polypeptide (e.g., SEQ ID NO: SEQ IDNO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90, or SEQ ID NO: 91) fused to a variant canine IgG-D Fcheterodimer chain 1 (e.g., SEQ ID NO: 14) and a glucagon polypeptide(e.g., SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95)fused to a variant canine IgG-D Fc heterodimer chain 2 (e.g., SEQ ID NO:15).

Example 5 GLP1 Fusion Proteins

The amino acid sequence of GLP1 is conserved among human, feline,canine, and equine, among other species. GLP1 proteolytic products(e.g., GLP1 (amino acids 7-37) (SEQ ID NO: 85) are rapidly degraded bydipeptidyl peptidase-4 (DPP-4) and are understood to have a short serumhalf-life of a couple minutes.

The design of contiguous polypeptides comprising at least one GLP1polypeptide and feline, canine, or equine IgG Fc polypeptides wasinvestigated to generate long-acting GLP1 fusion proteins. The followingconstructs were designed:

GLP1A-L1-Fc;  Formula (I):

Fc-L1-GLP1A; or  Formula (II):

GLP1A-L1-Fc-L2-GLP1B.  Formula (III):

wherein GLP1A is a first GLP1 polypeptide, GLP1B is a second GLP1polypeptide, L 1 and L2 are linkers; and Fc is an IgG Fc polypeptide ofa companion animal species.

GLP1 was modified to be DPP-4 resistant by replacing alanine with eitherglycine or serine at a position corresponding to position 8 of wild-typeGLP1 (7-37) (SEQ ID NO: 85). A minimal sequence of wild-type GLP1 (7-37)(SEQ ID NO:87) for binding to the N-terminal domain of mature felineGLP1R (SEQ ID NO: 49) was analyzed by three-dimensional protein modelingof the complex. Based on this modeling, the two C-terminal amino acidswere removed from the DPP-4 resistant GLP1 to generate GLP1-S8 (7-35)(SEQ ID NO: 86) and GLP1-G8 (7-35) (SEQ ID NO: 87) polypeptides.

GLP1 polypeptides when positioned at the C-terminus of a construct, suchas in formulas II and III, are not susceptible to DPP-4 degradation.Therefore, the alanine to glycine or serine substitution is notnecessary for GLP1 polypeptides positioned at the C-terminus.Accordingly, wild-type GLP1 (7-37) (SEQ ID NO: 85) may be used at theC-terminus.

The linker may be a flexible, non-structural linker, such as a glycine-and serine-rich linker. A flexible extension may be added to theC-terminus of the contiguous polypeptide. The extension may comprise aglycine residue (SEQ ID NO: 88), two glycine residues (SEQ ID NO: 89), athree glycine residues (SEQ ID NO: 90), four glycine residues (SEQ IDNO: 91), five glycine residues (SEQ ID NO: 92), six glycine residues(SEQ ID NO: 93), seven glycine residues (SEQ ID NO: 94), eight glycineresidues (SEQ ID NO: 95), or more glycine residues.

Example 6 GLP1 and Feline IgG Fc Fusion Proteins

Nucleotide sequences encoding (1) a contiguous polypeptide of Formula Ihaving a signal sequence, GLP1-G8 (7-35) (SEQ ID NO: 87), a flexiblelinker, and wildtype feline IgG2 Fc (ssGLP1-G8_I_WTfeIgG2; SEQ ID NO:96); and (2) a contiguous polypeptide of Formula III having a signalsequence, GLP1-G8 (7-35) (SEQ ID NO: 87), GLP1 (7-35) (SEQ ID NO: 89),two flexible linkers, a 2G C-terminal extension, and wildtype felineIgG2 Fc (ssGLP1-G8/GLP1-2G_III_WTfeIgG2; SEQ ID NO: 97) were synthesizedand cloned into separate mammalian expression vectors. Wild-type felineIgG2 Fc (SEQ ID NO: 16) was chosen based on its low or no C1Q bindingfor reduced complement activity and Protein A binding for ease ofpurification.

The resulting vectors were separately transfected to CHO cells. Thesupernatant containing the contiguous polypeptides without the signalpeptide (SEQ ID NOs: 24 and 23) was collected and filtered. Bothproteins were affinity purified using a Protein A column (CaptivA®Protein A Affinity Resin, Repligen). The proteins were determined to bemonomeric as assessed by HPLC gel filtration. However, the SDS-PAGEanalysis showed that a percentage of both continuous polypeptidesmigrated to the same position in the gel in the absence and presence ofreducing agent (DTT) (FIG. 2A). This result suggests that the hinge ofwild-type feline IgG2 Fc, which has only one pair of cysteine residues,may not be enough to form an effective disulfide linkage.

Three-dimensional protein modeling analysis of several ortholog hingestructures was used to determine the approximate locations for modifyingthe feline IgG2 hinge to increase disulfide formation. To increasedisulfide formation at the feline IgG2 hinge, the hinge sequence may bemodified by substituting an amino acid with cysteine. For example, avariant feline IgG2 Fc (SEQ ID NO: 17) having a modified hinge wasprepared by substituting Gly with Cys at an amino acid positioncorresponding to position 14 of SEQ ID NO: 16. The correspondingcontiguous polypeptides ssGLP1-G8_I_VARfeIgG2 (SEQ ID NO: 39) andssGLP1-G8/GLP1-2G_III_WTfeIgG2 (SEQ ID NO: 38) comprising variant felineIgG Fc of SEQ ID NO: 17 were designed, expressed in CHO cells, andpurified by Protein A chromatography. The amino acid sequences of thesecreted proteins after cleavage of the signal sequence are SEQ ID NOs26 and 25, respectively. The SDS-PAGE analysis of the variant felineIgG2 constructs showed a decrease in the amount of protein in the lowermolecular weight band in absence of reducing agent compared to thewild-type feline IgG2 constructs (compare FIG. 2 B to FIG. 2A). Theseresults suggest that the Fc covalent pairing was improved for bothvariant feline IgG2 constructs.

Furthermore, differential scanning fluorimetry was used to assess thestability of the contiguous polypeptides at various pH, as reflected bymean melting point temperature (n=3) (Table 9, below). The increasedstability of the variant feline IgG2 hinge is most evident at pH 6. Forexample, the constructs having variant feline IgG2 (SEQ ID NOs: 25 and26) exhibited a higher Tm at pH 6 (56.9 and 59.7° C.) than thecorresponding constructs having wild-type feline IgG2 (SEQ ID NOs: 23and 24), which had a Tm of 55.2 and 56.9° C., respectively.

TABLE 9 Mean Melting Point Temperature (Tm ° C.) (n = 3) Construct (10μg) pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 GLP1-G8/ NC NC NC 55.2 55.7 54.2GLP1-2G_III_WTfeIgG2 (SEQ ID NO: 23) GLP1-G8_I_WTfeIgG2 NC NC 48.5 56.959.9 59 (SEQ ID NO: 24) GLP1-G8/ NC NC NC 56.9 55 52.5GLP1-2G_III_WTfeIgG2 (SEQ ID NO: 25) GLP1-G8_I_VARfeIgG2 NC NC 53.1 59.759.9 58.2 (SEQ ID NO: 26) NC = no curve because no distinct transitionpoint was observed.

Contiguous polypeptides of Formulas I, II, and III comprising GLP1-S8(7-35) (SEQ ID NO: 86) instead of GLP1-G8 (7-35) (SEQ ID NO: 87) may besimilarly designed and prepared. For example, ssGLP1-S8_I_WTfeIgG2 (SEQID NO: 40), GLP1-S8_I_WTfeIgG2 (SEQ ID NO: 28),ssGLP1-S8/GLP1-3G_III_WTfeIgG2 (SEQ ID NO: 37), andGLP1-S8/GLP1-3G_III_WTfeIgG2 (SEQ ID NO: 27) may be prepared. Similarconstructs having variant instead of wild-type feline IgG Fc, such asGLP1-S8_I_VARfeIgG2, GLP1-S8_VARfeIgG2, GLP1-S8/GLP1-2G_III_VARfeIgG2,and GLP1-S8/GLP1-3G_III_VARfeIgG2, may also be prepared.

While feline IgG2 Fc was used in this example, contiguous polypeptidesof Formulas I, II, and III comprising feline IgG1a Fc or IgG1b Fcinstead of IgG2 may be designed and prepared. For example, similarcontiguous polypeptides having wild-type feline IgG1a Fc, wild-typefeline IgG1b Fc, variant feline IgG1a Fc, or variant feline IgG1b Fc(SEQ ID NOs: 80 to 83, respectively), may be designed and prepared.

Example 7 GLP1 and Canine IgG Fc Fusion Proteins

Various Formula I, II, and III contiguous polypeptides comprising avariant GLP1 and a canine IgG Fc may be designed and prepared. Forexample, a variant canine IgGD Fc (e.g., SEQ ID NO: 7 or SEQ ID NO: 62)may be chosen based on its low or no C1q binding for reduced complementactivity and Protein A binding for ease of purification. In addition, aflexible, non-structural linker, such as a glycine- and/or serine-richlinker, may be used.

GLP1-G8_I_VARcaIgGD (SEQ ID NO: 30) and GLP1-S8_I_VARcaIgGD (SEQ ID NO:32) are examples of Formula I contiguous polypeptides comprising (1)either GLP1-G8 (7-35) or GLP1-S8 (7-35), (2) a flexible linker, and (3)a variant canine IgGD Fc (e.g., SEQ ID NO: 7).GLP1-G8/GLP1-3G_III_VARcaIgGD (SEQ ID NO: 29) andGLP1-S8/GLP1-3G_III_VARcaIgGD (SEQ ID NO: 31) are examples of FormulaIII contiguous polypeptides comprising (1) either GLP1-G8 (SEQ ID NO:87) or GLP1-S8 (SEQ ID NO: 86), (2) GLP1 (7-35) (SEQ ID NO: 61), (3) twoflexible linkers, (4) a 3G C-terminal extension, and (5) a variantcanine IgGD Fc.

The contiguous polypeptides may be designed with a signal sequence, adifferent GLP1 polypeptide (e.g., SEQ ID NO: 85, 86, 87, 98, or 99), ordifferent modifications to the canine IgG Fc. Examples with suchvariations include ssGLP1-S8/GLP1-2G_III_VARcaIgGD (SEQ ID NO: 41),ssGLP1-G8/GLP1-2G_III_VARcaIgGD (SEQ ID NO: 42),ssGLP1-S8/GLP1-3G_III_VARcaIgGD (SEQ ID NO: 43),GLP1-S8/GLP1-2G_III_VARcaIgGD (SEQ ID NO: 105), andGLP1-G8/GLP1-2G_III_VARcaIgGD (SEQ ID NO: 106).

Furthermore, contiguous polypeptides of Formulas I, II, and III maycomprise a wild-type canine IgGD, or a wild-type or variant canine IgGA,IgGB, or IgGC, instead of a variant canine IgGD. For example, similarcontiguous polypeptides may be designed and prepared having a wild-typecanine IgGA Fc, IgGB Fc, IgGC Fc, or IgGD Fc (e.g., SEQ ID NO: 1, 2, 3,or 4, respectively). Additional examples include contiguous polypeptidescomprising a variant canine IgGA Fc (e.g., SEQ ID NO: 5 or 60), avariant canine IgGB Fc (e.g., SEQ ID NO: 78), or a variant canine IgGCFc (e.g., SEQ ID NO: 6, 61, 79, or 84).

GLP1-G8/GLP1-3G_III_VARcaIgGD (SEQ ID NO: 29) and GLP1-G8_I_VARcaIgGD(SEQ ID NO: 30) were expressed separately in CHO cells and thesupernatants containing the proteins collected and filtered. Bothcontiguous polypeptides were affinity purified by Protein Achromatography. The SDS-PAGE profiles of the two contiguous polypeptidesin the absence and presence of reducing agent (DTT) were compared andthe results suggested that the Fc disulfide bond was effectively formedfor both polypeptides (data not shown).

Example 8 GLP1 and Equine IgG Fc Fusion Proteins

Various Formula I, II, and III contiguous polypeptides comprising avariant GLP1 and an equine IgG Fc may be designed and prepared. Forexample, a variant canine IgG2 Fc (e.g., SEQ ID NO: 19, 71, or 72) maybe chosen based on its low or no C1q binding for reduced complementactivity and Protein A binding for ease of purification. In addition, aflexible, non-structural linker, such as a glycine- and/or serine-richlinker, may be used.

GLP1-G8_I_VAReqIgG2 (SEQ ID NO: 34) and GLP1-S8_I_VAReqIgG2 (SEQ ID NO:36) are examples of Formula I contiguous polypeptides comprising (1)either GLP1-G8 or GLP1-S8, (2) a flexible linker, and (3) a variantequine IgG2 Fc (e.g., SEQ ID NO: 19). GLP1-G8/GLP1-3G_III_VAReqIgG2 (SEQID NO: 33) and GLP1-S8/GLP1-3G_III_VAReqIgG2 (SEQ ID NO: 35) areexamples of Formula III contiguous polypeptides comprising (1) eitherGLP1-G8 (7-35) (SEQ ID NO: 87) or GLP1-S8 (7-35) (SEQ ID NO: 86), (2)GLP1 (7-35) (SEQ ID NO: 61), (3) two flexible linkers, (4) a 3GC-terminal extension, and (5) a variant equine IgGD Fc.

The contiguous polypeptides may be designed with a signal sequence, adifferent GLP1 analog (e.g., SEQ ID NO: 86, 87, 98, or 99), a glycineextension (e.g., SEQ ID NO: 88 to 95) or additional modifications to theequine IgG Fc. Examples with such variations includessGLP1-G8/GLP1-3G_III_VAReqIgG2 (SEQ ID NO: 44), ssGLP1-G8_I_VAReqIgG2(SEQ ID NO: 45), ssGLP1-S8/GLP1-3G_III_VAReqIgG2 (SEQ ID NO: 46), andssGLP1-S8_I_VAReqIgG2 (SEQ ID NO: 47).

Furthermore, contiguous polypeptides of Formulas I, II, and III maycomprise a wild-type equine IgG2, or a wild-type or variant equine IgG1,IgG3, IgG4, IgG5, IgG6, IgG7, instead of a variant equine IgG2. Forexample, similar contiguous polypeptides may be designed and preparedhaving a wild-type equine IgG1 Fc (e.g., SEQ ID NO: 63), IgG2 Fc (e.g.,SEQ ID NO: 18 or 64), IgG3 Fc (e.g., SEQ ID NO: 65), IgG4 Fc (e.g., SEQID NO: 66), IgG5 Fc (e.g., SEQ ID NO: 67), IgG6 Fc (e.g., SEQ ID NO:68), or IgG7 Fc (e.g., SEQ ID NO: 69). Additional examples includecontiguous polypeptides comprising a variant equine IgG1Fc (e.g., SEQ IDNO: 70), a variant equine IgG3 Fc (e.g., SEQ ID NO: 73), a variantequine IgG4 Fc (e.g., SEQ ID NO: 74), a variant equine IgG5 Fc (e.g.,SEQ ID NO: 75), a variant equine IgG6 Fc (e.g., SEQ ID NO: 76), or avariant equine IgG7 Gc (e.g., SEQ ID NO: 77).

The nucleotide sequences encoding ssGLP1-G8/GLP1-3G_III_VAReqIgG2 (SEQID NO: 44) and ssGLP1-G8_I_VAReqIgG2 (SEQ ID NO: 45) were synthesizedand cloned into separate mammalian expression vectors. The resultingvectors were separately transfected to CHO cells. The supernatantcontaining the contiguous polypeptides following cleavage of the signalpeptide (SEQ ID NOs: 103 and 104) was collected and filtered. Bothproteins were affinity purified using a Protein A column (CaptivA®Protein A Affinity Resin, Repligen). were expressed separately in CHOcells and the supernatants containing the proteins collected andfiltered. Both contiguous polypeptides were affinity purified by ProteinA chromatography. The SDS-PAGE profiles of the two contiguouspolypeptides in the absence and presence of reducing agent (DTT) werecompared and the results suggested that the Fc disulfide bond waseffectively formed for both polypeptides (data not shown).

Example 9 Expression and Purification of Feline GLP1R N-Terminal SolubleDomain

The N-terminal domain of mature feline GLP1R (SEQ ID NO: 49) responsiblefor binding GLP1 to GLP1R was identified from the full-length felineGLP1R amino acid sequence (SEQ ID NO: 48). Nucleotide sequences encoding(1) a signal sequence, feline N-terminal GLP1R, human Fc, and a poly-Histag (ssFeGLP1R-N-huFc_PolyHis; SEQ ID NO: 50) and (2) a signal sequence,feline N-terminal GLP1R, and a poly-His tag (ssFeGLP1R-N_polyHis; SEQ IDNO: 51) were synthesized and cloned into separate mammalian expressionvectors. The resulting vectors were separately transfected to CHO cells.The supernatant containing the polypeptides was collected and filtered.The proteins were affinity purified using a Ni-Sepharose column orProtein A column (CaptivA® Protein A Affinity Resin, Repligen) for thehuFc construct. Both proteins were used for GLP1 functional binding andELISAs.

Example 10 GLP1 Fusion Protein Binding Kinetics

The binding affinity of GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) toFeGLP1R-N-huFc_PolyHis was assessed using biolayer interferometry(Octet). Briefly, FeGLP1R-N-huFc_PolyHis was biotinylated, the freeunreacted biotin was removed, and the biotinylated protein was capturedto streptavidin sensor tips. The association of different concentrationsGLP1-G8_I_VARfeIgG2 was monitored for ninety seconds. Dissociation wasmonitored for 600 seconds. A buffer only blank curve was subtracted tocorrect for any drift. The data were fit to a 1:1 binding model usingForteBio™ data analysis software to determine the k_(on), k_(off), andthe K_(d). The buffer for dilutions and all binding steps was: 20 mMphosphate, 150 mM NaCl, pH 7.2. The K_(d) between Feline GLP1RN-terminaldomain and GLP1-G8_I_VARfeIgG2 was between 8.0×10⁻⁹ and 16×10⁻⁹ M.

Example 11 GLP1 Fusion Protein Bioactivity in a Cell-Based Assay

CHOK-1-GL1R cell line (Discoverx, cat #95-0062C2), a hamster ovariancell line which overexpresses Gs-coupled human glucagon ligand peptide-1receptors (GLP1R) on the cell surface, was used to measure GLP1 cellularactivity with a cAMP Hunter Bioassay kit (Discoverx, Cat #95-0062Y2).Cells were plated in a 96-well plate and incubated at 37° C., 5% CO₂ for24 hours. Cells were then treated with a control agonist—either GLP1human (37 a.a.) (Prospec, Cat #HOR-236) or Extendin-4 (Discoverx, Cat#92-1115)—or a contiguous polypeptide comprising a variant GLP1 at aseries of 3-fold dilutions followed by incubation at 37° C. for 30 min.The contiguous polypeptides tested were GLP1-G8/GLP1-2G_III_VARfeIgG2(SEQ ID NO: 25) and GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26).

Upon GLP1 binding to the Gs-coupled GLP1R receptor, Gs stimulatesadenylate cyclase to generate cAMP. At the end of incubation, cAMPAntibody Reagent and cAMP Working Detection Solution, which containslysis buffer, β-galactosidase (β-gal) small fragment conjugated cAMPs,and substrates, were added to the cells. The cells were incubated in thedark for 1 hour at room temperature to allow the immunocompetitionreaction to occur between endogenously generated cAMPs and β-gal smallfragment conjugated cAMPs for cAMP antibody binding.

At the end of the 1 hour incubation, cAMP Solution A containing β-gallarge fragments, which can complement with the free (non-antibodybinding) β-gal small fragment-cAMPs to form functional enzymes, wereadded to the cell lysate. The lysate was incubated for 3 to 6 hours inthe dark at room temperature to allow the β-gal to hydrolyze thesubstrate and generate luminescent signals.

The more unbound free β-gal small fragment-cAMPs that remain, the morefunctional β-gal enzymes form. Therefore, the amount of signal producedis directly proportional to the amount of cAMP in the cell lysate. Atthe end of incubation, luminescence was read on a Synergy HT microplatereader (Biotek, Winooski, Vt.). The EC50s were calculated with asoftware GraphPad Prism (GraphPad Software, Inc. La Jolla, USA).

FIG. 3 shows a plot of the relative light units (RLU) versusconcentration for each sample tested. The EC50s are listed in Table 10,below. Both GLP1-G8/GLP1-2G_III_VARfeIgG2 (SEQ ID NO: 25) andGLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) are more active than GLP1 (7-37) andExtendin-4. GLP1-G8/GLP1-2G_III_VARfeIgG2 (SEQ ID NO: 25) is more potentthan GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26), suggesting that the C-terminalGLP1 may contribute additional activity.

TABLE 10 Sample EC 50 (nM) GLP1 (7-37) 0.1449 Extendin-4 0.4596GLP1-G8/GLP1-2G_III_VARfeIgG2 0.01751 (SEQ ID NO: 25)GLP1-G8_I_VARfeIgG2 0.02806 (SEQ ID NO: 26)

Example 12 GLP1 Fusion Protein Long-Term Stability

GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was stored in PBS, pH7.2 at aconcentration of 1.3 mg/mL, placed in 1.5 mL Eppendorf tube, and storedat 2-8° C. for one year (Lot 2-29-2016). GLP1-G8_IVARfeIgG2 (SEQ ID NO:26) was also stored in PBS, pH7.2 at a concentration of 10 mg/mL, placedin 1.5 mL Eppendorf tube, and stored at 2-8° C. for one day (Lot2-2-2017). To evaluate stability, the stored sample were analyzed bycell-based assay using the same CHOK-1-GL1R cell line (Discoverx, cat#95-0062C2) described in Example 11, but cellular activity was assessedusing a cAMP-Glo™ Max Assay (Promega, Cat #PAV1682).

CHOK-1-GL1R cells were plated in a 96-well plate (Corning, Cat #3610) ata density of 20,000 cells per well in F-12K Medium (ATCC, Cat #ATCC®30-2004) supplemented with 10% Fetal Bovine Serum, heat inactivated(Sigma, Cat #2868) and incubated at 37° C., 5% CO2 for 24 hours. Cellswere then stimulated with a control agonist GLP1 human (37 a.a.)(Prospec, Cat #HOR-236), Lot 2-20-2016, or Lot 2-2-2017 at a series of3-fold dilutions with serum-free medium followed by addition of CompleteInduction Buffer which contains MgCl₂ to a final concentration 20 mM,isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich Cat.#17018) to a finalconcentration 500 μM and Ro 20-1724 [4-(3-butoxy-4-methoxy-benzyl)imidazolidone] (Sigma Aldrich, Cat.#B8279) to a final concentration 100μL.

The cells were incubated at room temperature for 30 minutes. In thisprocess, upon GLP1 binding to the Gs-coupled GLP1R receptor, Gsstimulates adenylate cyclase to generate cAMP. At the end of incubation,cAMP Detection Solution, which contains an inactive protein kinase Aholoenzyme, protein kinase A substrate, and lysis buffer, was added tothe cells. The plates placed on an orbital shaker for 1-2 minutes andthen incubated at room temperature (23° C.) for 20 minutes. CellularcAMP will activate protein kinase A by binding its regulatory-inhibitorysubunits and releasing the catalytic subunits. The free catalyticsubunits catalyze the transfer of the terminal phosphate of ATP to theprotein kinase A substrate, consuming cellular ATP in the process. Atthe end of incubation, a luciferase-based Kinase-Glo® Reagent was addedto the cell lysates and the plates were shaken on an orbital shaker for2 min followed by incubation in the dark at room temperature for 10 min.

Mono-oxygenation of luciferin was catalyzed by luciferase in thepresence of Mg²⁺ and ATP that presented in the cell lysate, resulting ina luminescent signal proportional to the amount of ATP in the cells. Atthe end of 10 min incubation, the plate was read on a Synergy HTmicroplate reader (Biotek, Winooski, Vt.). Luminescence is proportionalto ATP levels but inversely proportional to cAMP levels. Thus, as cAMPconcentration increases, luminescence decreases.

FIG. 4 shows the results of the cell-based assay as a plot ofluminescence versus concentration for Lot 2-29-2016 compared to Lot2-2-2017 and GLP1 (31 a.a.). The GLP1-G8_I_VARfeIgG2 sample maintainedcellular activity after storage in PBS at 2-8° C. for one year.

Example 13 GLP1 Fusion Protein Serum Stability

GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was stored in PBS, pH7.2 with felineserum at 37° C. for 24 hours to test in vitro serum stability. Thecell-based assay was performed as described in Example 12 and resultssuggested that the activity was maintained (data not shown). Inaddition, no visible degradations were observed by Western blot analysis(FIG. 5).

Example 14 GLP1 Fusion Protein In Vivo Pharmacokinetics

GLP1-G8_I_VARfeIgG2 (SEQ ID NO: 26) was administered as a single dose (2mg/kg) by subcutaneous injection to 5 cats. Serum samples were takenbefore dosing (time 0) and at 4 hours, 8 hours, 12 hours, 24 hours, 48hours, 72 hours, and 168 hours. The concentration of GLP1-G8_I_VARfeIgG2in the serum samples was measured by quantitative ELISA.GLP1-G8_I_VARfeIgG2 polypeptide with a detection limit of 4 ng/mL wasused as a reference. The serum concentration of GLP1-G8_I_VARfeIgG2 wasplotted against time (FIG. 6). The mean serum half-life (t_(1/2)) ofGLP1-G8_I_VARfeIgG2 was 39 hours. The average T_(max) was 22 hours, theaverage C_(max) was 12 μg/mL, and the mean area under the curve (AUC)was about 950 m(h)/mL.

The quantitative ELISA used an anti-GLP1 antibody (4F3, NovusBiologicals, Catalog No. NBP1-97413) and a goat anti-cat IgG-Fc, HRPconjugated antibody (Bethyl Laboratories, Inc., Catalog No. A20-117P)for quantification of GLP1-G8_I_VARfeIgG2 in feline serum samples fromthe in vivo pharmacokinetics study. A 96-well plate was coated withanti-GLP1 antibody (5 μg/mL in coating buffer, 100 μl/well). The platewas sealed and incubated overnight at 4° C. The plate was washed intriplicate with 1×TBST (10×TBST, Teknova, Catalog No. T9511) andblocking buffer was added. After removing the blocking buffer, serialdilutions of reference standard and samples in blocking buffer wereadded (100 μl/well) and the plate was incubated for 2 hours at roomtemperature. The plate was washed in triplicate with 1×TBST and goatanti-cat IgG Fc antibody was added (0.1 μg/mL in blocking buffer, 100μl/well). After incubation for 1 hour at room temperature, the plate waswashed 5 times with 1×TBST. TMB substrate (ScyTek, Catalog No. TM1999)was added (100 μl/well) and allowed to incubate at room temperature for1 minute. The reaction was stopped by the addition of 2M H₂SO₄ (50μl/well). Absorbance at 450 nm was measured and the concentration ofGLP1-G8_I_VARfeIgG2 in the serum samples calculated.

Furthermore, GLP1-G8_I_VARfeIgG2 concentration in the same serum samples(no DPP-4 inhibitor added) was assessed using a cell-based activityassay. The same CHOK-1-GL1R cell line (Discoverx, cat #95-0062C2) andcAMP-Glo™ Max Assay (Promega, Cat #PAV1682) described in Example 12 wasused. In this Example, however, the cells were stimulated with a controlagonist GLP1 human (37 a.a.) (Prospec, Cat #HOR-236) or samples of catserum taken before dosing (time 0) and at at 4 hours, 8 hours, 12 hours,24 hours, 48 hours, 72 hours, and 168 hours diluted in serum-free medium(5%, 0.5%, and 0.05% dilutions). The concentration ofGLP1-G8_I_VARfeIgG2 in each sample was calculated using SoftMax pro 7(Molecular Devices, Sunnyvale, Calif.). The mean concentration ofGLP1-G8_I_VARfeIgG2 for the 5 cats was plotted against time (FIG. 7).The mean AUC was about 840 μg(h)/mL and the mean t_(1/2) was 36 hours.The concentrations calculated from the cell-based activity assay areconsistent with the concentrations obtained from the ELISA, whichsuggests that the variant GLP1 detected by ELISA is biologically active.

Example 15 GLP1, Glucagon, and IgG Fc Fusion Proteins

To investigate a long acting GLP1 receptor and Glucagon receptor dualagonist, contiguous polypeptides comprising a GLP1 polypeptide, aglucagon polypeptide, and an IgG Fc polypeptide having the followingconstructs were designed:

GLP1-L1-Fc-L2-Gluc; and  Formula (IV):

Gluc-L1-Fc-L2-GLP1,  Formula (V):

wherein GLP1 is a GLP1 polypeptide, Gluc is a glucagon polypeptide, L1and L2 are linkers, and Fc is an Fc polypeptide.

As discussed above, GLP1 was modified to be DPP-4 resistant by replacingalanine with either glycine or serine at a position corresponding toposition 8 of wild-type GLP1 (7-37) (SEQ ID NO: 85). In addition, theDPP-4 resistant GLP1 was further modified by removing the two C-terminalamino acids to generate variant GLP1-S8 (7-35) (SEQ ID NO: 86) andvariant GLP1-G8 (7-35) (SEQ ID NO: 87) polypeptides.

GLP1 polypeptides when positioned at the C-terminus of a construct, suchas in Formula (V), are not susceptible to DPP-4 degradation. Therefore,the alanine to glycine or serine substitution is not necessary for GLP1polypeptides positioned at the C-terminus. Accordingly, wild-type GLP1(7-37) (SEQ ID NO: 85) may be used at the C-terminus.

The linker may be a flexible, non-structural linker, such as a glycine-and serine-rich linker. A flexible extension may be added to theC-terminus of the contiguous polypeptide. The extension may comprise aglycine residue (SEQ ID NO: 88), two glycine residues (SEQ ID NO: 89), athree glycine residues (SEQ ID NO: 90), four glycine residues (SEQ IDNO: 91), five glycine residues (SEQ ID NO: 92), six glycine residues(SEQ ID NO: 93), seven glycine residues (SEQ ID NO: 94), eight glycineresidues (SEQ ID NO: 95), or more glycine residues.

The contiguous polypeptide may comprise a wildtype glucagon polypeptide(e.g., SEQ ID NO: 21) or a variant glucagon polypeptide.

The contiguous polypeptide may comprise a human IgG Fc or an IgG Fc of acompanion animal species, such as canine, feline, or equine. The subtypeof IgG Fc used may be based on having low or no C1q binding activityand/or having Protein A binding capacity. For example, a wild-type orvariant human, canine, equine, or feline IgG Fc having low or no C1qbinding and/or having Protein A binding capacity may be used (e.g., SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, or 84).

Exemplary contiguous polypeptides comprising a GLP1 polypeptide, aGlucagon polypeptide, and a feline IgG Fc polypeptide includeGLP1-G8/Gluc-3G_IV_WTfeIgG2 (SEQ ID NO: 52) and Gluc/GLP1-2G_V_WTfeIgG2(SEQ ID NO: 53).

Exemplary contiguous polypeptides comprising a GLP1 polypeptide, aGlucagon polypeptide, and a canine IgG Fc polypeptide includeGLP1-G8/Gluc-4G_IV_VARcaIgGD (SEQ ID NO: 54) andGluc/GLP1-3G_V_VARcaIgGD (SEQ ID NO: 55).

Exemplary contiguous polypeptides comprising a GLP1 polypeptide, aGlucagon polypeptide, and an equine IgG Fc polypeptide includeGLP1-G8/Gluc-4G_IV_VAReqIgGD (SEQ ID NO: 56) andGluc/GLP1-3G_V_VAReqIgG2 (SEQ ID NO: 57).

Exemplary contiguous polypeptides comprising a GLP1 polypeptide, aGlucagon polypeptide, and a human IgG Fc polypeptide includeGLP1-G8/Gluc-4G_IV_huIgG4 (SEQ ID NO: 8) and Gluc/GLP1-3G_V_huIgG4 (SEQID NO: 59).

Example 16 Variant IgG Fc Polypeptides for Enhanced Hinge DisulfideFormation

Additional three-dimensional protein modeling analysis of severalortholog hinge structures was used to modify feline and equine IgGhinges to enhance disulfide formation. To enhance disulfide formation atthe feline IgG hinge, the hinge sequence may be modified by substitutinglysine with proline at a position corresponding to position 16 of felineIgG2 (SEQ ID NO: 16), of feline IgG1a (SEQ ID NO: 80 or SEQ ID NO: 117),or of feline IgG1b (SEQ ID NO: 81 or SEQ ID NO: 118) (e.g., K16P).Examples of amino acid sequences of variant feline IgG polypeptideshaving a modified hinge include SEQ ID NO: 125, SEQ ID NO: 126, and SEQID NO: 127.

To enhance disulfide formation at the equine IgG hinge, the hingesequence may be modified by substitution cysteine with serine at aposition corresponding to position 3 of an equine IgG (e.g., IgG2 Fc(SEQ ID NO: 129)) and/or substituting glutamine with proline at aposition corresponding to position 20 of an equine IgG (e.g., IgG2 Fc(SEQ ID NO: 129) (e.g., C3S, Q20P). Examples of amino acid sequences ofvariant equine IgG polypeptides having a modified hinge include SEQ IDNO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 134, and SEQ ID NO:135.

Example 17 Variant IgG Fc Polypeptides for Enhanced RecombinantProduction and/or Enhanced Hinge Disulfide Formation

Three-dimensional protein modeling was used to design feline and equinevariant IgG Fc polypeptides comprising sequences from the hinge regionfrom a different IgG isotype for enhanced recombinant production andimproved hinge disulfide formation. Variant feline IgG2 Fc polypeptidesmay be prepared that comprise sequences from the hinge region of felineIgG1a or IgG1b (e.g., SEQ ID NO: 125). In addition, variant equine IgG2Fc polypeptides may be prepared that comprise sequences from the hingeregion of equine IgG1 (e.g., SEQ ID NO: 19).

Levels of recombinant production of variant IgG Fc polypeptides and/orlevels of hinge disculfide formation may be determined and compared tothat of another IgG Fc by SDS-PAGE analysis under reducing andnon-reducing conditions (e.g., the corresponding wild-type IgG Fc of thesame or different isotype, or a wild-type or variant IgG Fc of anothercompanion animal, etc.).

1. A polypeptide comprising a variant IgG Fc polypeptide comprising atleast one amino acid modification relative to a wild-type IgG Fcpolypeptide of a companion animal species, wherein the variant IgG Fcpolypeptide has increased binding affinity to Protein A relative to thewild-type IgG Fc polypeptide.
 2. A polypeptide comprising a variant IgGFc polypeptide comprising at least one amino acid modification relativeto a wild-type IgG Fc polypeptide of a companion animal species, whereinthe variant IgG Fc polypeptide has reduced binding affinity to C1qand/or CD16 relative to the wild-type IgG Fc polypeptide.
 3. Thepolypeptide of claim 1 or claim 2, wherein the variant IgG Fcpolypeptide binds to C1q and/or CD16 with a dissociation constant(K_(d)) of greater than 5×10⁻⁶ M, greater than 1×10⁻⁵ M, greater than5×10⁻⁵ M, greater than 1×10⁻⁴ M, greater than 5×10⁻⁴ M, or greater than1×10⁻³ M, as measured by biolayer interferometry.
 4. The polypeptide ofany one of the preceding claims, wherein the variant IgG Fc polypeptidehas increased binding affinity to Protein A relative to the wild-typeIgG Fc polypeptide.
 5. The polypeptide of any one of the precedingclaims, wherein the variant IgG Fc polypeptide binds to Protein A with adissociation constant (K_(d)) of less than 5×10′ M, less than 1×10⁻⁶ M,less than 5×10⁻⁷ M, less than 1×10⁻⁷ M, less than 5×10⁻⁸ M, less than1×10⁻⁸ M, less than 5×10⁻⁹ M, less than 1×10⁻⁹ M, less than 5×10⁻¹⁰ M,less than 1×10⁻¹⁰ M, less than 5×10⁻¹¹ M, less than 1×10⁻¹¹ M, less than5×10⁻¹² M, or less than 1×10⁻¹² M, as measured by biolayerinterferometry.
 6. The polypeptide of any one of the preceding claims,wherein the companion animal species is canine, feline, or equine. 7.The polypeptide of any one of the preceding claims, wherein thewild-type IgG Fc polypeptide is a) a canine IgG-A Fc, IgG-B Fc, IgG-CFc, or IgG-D Fc; b) an equine IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, IgG5Fc, IgG6 Fc, or IgG7 Fc; or c) a feline IgG1a Fc, IgG1b Fc, or IgG2 Fc.8. A polypeptide comprising a variant IgG Fc polypeptide comprising atleast one amino acid modification to a hinge region relative to awild-type feline or equine IgG Fc polypeptide, wherein the variant IgGFc polypeptide has increased recombinant production and/or increasedhinge disulfide formation relative to the wild-type IgG Fc polypeptide,as determined by SDS-PAGE analysis under reducing and/or nonreducingconditions.
 9. The polypeptide of any one the preceding claims, whereinthe variant IgG Fc polypeptide comprises: a) at least one amino acidsubstitution relative to a wild-type feline IgG Fc polypeptide, whereinthe variant IgG Fc polypeptide comprises an amino acid substitution at aposition corresponding to position 16 of SEQ ID NO: 16, SEQ ID NO: 80,SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; b) at least one aminoacid substitution relative to a wild-type equine IgG Fc polypeptide,wherein the variant IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 3 of SEQ ID NO:129; and/or c) at least one amino acid substitution relative to awild-type equine IgG Fc polypeptide, wherein the variant IgG Fcpolypeptide comprises an amino acid substitution at a positioncorresponding to position 20 of SEQ ID NO:
 129. 10. The polypeptide ofany one the preceding claims, wherein the variant IgG Fc polypeptidecomprises: a) at least one amino acid substitution relative to awild-type feline IgG Fc polypeptide, wherein the variant IgG Fcpolypeptide comprises an amino acid substitution at position 16 of SEQID NO: 16, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 117, or SEQ ID NO:118; b) at least one amino acid substitution relative to a wild-typeequine IgG Fc polypeptide, wherein the variant IgG Fc polypeptidecomprises an amino acid substitution at position 3 of SEQ ID NO: 129;and/or c) at least one amino acid substitution relative to a wild-typeequine IgG Fc polypeptide, wherein the variant IgG Fc polypeptidecomprises an amino acid substitution at position 20 of SEQ ID NO: 129.11. The polypeptide of any one the preceding claims, wherein the variantIgG Fc polypeptide comprises: a) at least one amino acid substitutionrelative to a wild-type feline IgG Fc polypeptide, wherein the variantIgG Fc polypeptide comprises a proline at a position corresponding toposition 16 or at position 16 of SEQ ID NO: 16, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 117, or SEQ ID NO: 118; b) at least one amino acidsubstitution relative to a wild-type equine IgG Fc polypeptide, whereinthe variant IgG Fc polypeptide comprises a serine at a positioncorresponding to position 3 or at position 3 of SEQ ID NO: 129; and/orc) at least one amino acid substitution relative to a wild-type equineIgG Fc polypeptide, wherein the variant IgG Fc polypeptide comprises aproline at a position corresponding to position 20 or at position 20 ofSEQ ID NO:
 129. 12. The polypeptide of any one the preceding claims,wherein the variant IgG Fc polypeptide comprises a hinge region or aportion of a hinge region from an IgG Fc polypeptide of a differentisotype.
 13. The polypeptide of any one of the preceding claims, whereinthe variant IgG Fc polypeptide comprises a hinge region or a portion ofa hinge region from a wild-type feline IgG-1a Fc polypeptide, from awild-type feline IgG-1b Fc polypeptide, or from a wild-type equine IgG1Fc polypeptide.
 14. The polypeptide of any one of the preceding claims,wherein the variant IgG Fc polypeptide comprises SEQ ID NO: 19, SEQ IDNO: 125 or SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ IDNO: 133, SEQ ID N: 134, SEQ ID NO:
 135. 15. A polypeptide comprising anamino acid sequence of SEQ ID NO: 19, SEQ ID NO: 125 or SEQ ID NO: 126,SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: SEQ ID NO: 129, SEQ ID NO:130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID N: 134, SEQID NO:
 135. 16. A polypeptide comprising a variant IgG2 Fc polypeptidecomprising at least one amino acid substitution relative to a wild-typefeline IgG2 Fc polypeptide, wherein the at least one amino acidsubstitution is a cysteine, and wherein the variant IgG2 Fc polypeptideis capable of forming at least one additional inter-chain disulfidelinkage relative to the wild-type feline IgG2 Fc polypeptide.
 17. Thepolypeptide of any one of the preceding claims, wherein the variant IgGFc polypeptide comprises at least one amino acid substitution relativeto a wild-type feline IgG Fc polypeptide, wherein the at least one aminoacid substitution is a cysteine, and wherein the variant IgG Fcpolypeptide is capable of forming at least one additional inter-chaindisulfide linkage relative to the wild-type feline IgG Fc polypeptide.18. The polypeptide of any one of the preceding claims, wherein thevariant IgG Fc polypeptide comprises a cysteine at a positioncorresponding to position 8, position 9, position 10, position 11,position 12, position 13, position 14, position 15, or position 16 ofSEQ ID NO:
 16. 19. The polypeptide of any one of the preceding claims,wherein the variant IgG Fc polypeptide comprises a cysteine at aposition corresponding to position 14 of SEQ ID NO:
 16. 20. Thepolypeptide of any one of the preceding claims, wherein the variant IgGFc polypeptide comprises a cysteine at position 14 of SEQ ID NO:
 16. 21.The polypeptide of any one of the preceding claims, wherein the variantIgG Fc polypeptide is at least 90% identical, at least 95% identical, atleast 97% identical, or at least 99% identical to the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO:83, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 107, SEQ ID NO: 108, SEQID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ IDNO:122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126,SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ IDNO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135,SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 140, SEQ IDNO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145,SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ IDNO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154,SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO:
 157. 22. The polypeptideof any one of the preceding claims, wherein the variant IgG Fcpolypeptide comprises SEQ ID NO:
 17. 23. A polypeptide comprising anamino acid sequence of SEQ ID NO:
 17. 24. The polypeptide of any one ofthe preceding claims, wherein the variant IgG Fc polypeptide comprises:a) an amino acid substitution at a position corresponding to position 21of SEQ ID NO: 1, an amino acid substitution at a position correspondingto position 23 of SEQ ID NO: 1, an amino acid substitution at a positioncorresponding to position 25 of SEQ ID NO: 1, an amino acid substitutionat a position corresponding to position 80 of SEQ ID NO: 1, an aminoacid substitution at a position corresponding to position 205 of SEQ IDNO: 1, and/or an amino acid substitution at a position corresponding toposition 207 of SEQ ID NO: 1; b) an amino acid substitution at aposition corresponding to position 21 of SEQ ID NO: 3, an amino acidsubstitution at a position corresponding to position 23 of SEQ ID NO: 3,and/or an amino acid substitution at a position corresponding toposition 24 of SEQ ID NO: 3; c) an amino acid substitution at a positioncorresponding to position 21 of SEQ ID NO: 4, an amino acid substitutionat a position corresponding to position 23 of SEQ ID NO: 4, an aminoacid substitution at a position corresponding to position 25 of SEQ IDNO: 4, an amino acid substitution at a position corresponding toposition 80 of SEQ ID NO: 4, and/or an amino acid substitution at aposition corresponding to position 207 of SEQ ID NO: 4; d) an amino acidsubstitution at a position corresponding to position 15 of SEQ ID NO:64, and/or an amino acid substitution at a position corresponding toposition 203 of SEQ ID NO: 64; e) an amino acid substitution at aposition corresponding to position 199 of SEQ ID NO: 67, and/or an aminoacid substitution at a position corresponding to position 200 of SEQ IDNO: 67; and/or f) an amino acid substitution at a position correspondingto position 199 of SEQ ID NO: 68, an amino acid substitution at aposition corresponding to position 200 of SEQ ID NO: 68, an amino acidsubstitution at a position corresponding to position 201 of SEQ ID NO:68, and/or an amino acid substitution at a position corresponding toposition 202 of SEQ ID NO:
 68. 25. The polypeptide of any one of thepreceding claims, wherein the variant IgG Fc polypeptide comprises: a)an amino acid substitution at position 21 of SEQ ID NO: 1, an amino acidsubstitution at position 23 of SEQ ID NO: 1, an amino acid substitutionat position 25 of SEQ ID NO: 1, an amino acid substitution at position80 of SEQ ID NO: 1, an amino acid substitution at position 205 of SEQ IDNO: 1, and/or an amino acid substitution at position 207 of SEQ ID NO:1; b) an amino acid substitution at position 21 of SEQ ID NO: 3, anamino acid substitution at position 23 of SEQ ID NO: 3, and/or an aminoacid substitution at position 24 of SEQ ID NO: 3; c) an amino acidsubstitution at position 21 of SEQ ID NO: 4, an amino acid substitutionat position 23 of SEQ ID NO: 4, an amino acid substitution at position25 of SEQ ID NO: 4, an amino acid substitution at position 80 of SEQ IDNO: 4, and/or an amino acid substitution at position 207 of SEQ ID NO:4; d) an amino acid substitution at position 15 of SEQ ID NO: 64, and/oran amino acid substitution at position 203 of SEQ ID NO: 64; e) an aminoacid substitution at position 199 of SEQ ID NO: 67, and/or an amino acidsubstitution at position 200 of SEQ ID NO: 67; and/or f) an amino acidsubstitution at position 199 of SEQ ID NO: 68, an amino acidsubstitution at position 200 of SEQ ID NO: 68, an amino acidsubstitution at position 201 of SEQ ID NO: 68, and/or an amino acidsubstitution at position 202 of SEQ ID NO:
 68. 26. The polypeptide ofany one of the preceding claims, wherein the variant IgG Fc polypeptidecomprises: a) a threonine at a position corresponding to position 21 ofSEQ ID NO: 1, a leucine at a position corresponding to position 23 ofSEQ ID NO: 1, an alanine at a position corresponding to position 25 ofSEQ ID NO: 1, a glycine at a position corresponding to position 80 ofSEQ ID NO: 1, an alanine at a position corresponding to position 205 ofSEQ ID NO: 1, and/or a histidine at a position corresponding to position207 of SEQ ID NO: 1; b) a threonine at a position corresponding toposition 21 of SEQ ID NO: 3, a leucine at a position corresponding toposition 23 of SEQ ID NO: 3, and/or an isoleucine at a positioncorresponding to position 24 of SEQ ID NO: 3; c) a threonine at aposition corresponding to position 21 of SEQ ID NO: 4, a leucine at aposition corresponding to position 23 of SEQ ID NO: 4, an alanine at aposition corresponding to position 25 of SEQ ID NO: 4, a glycine at aposition corresponding to position 80 of SEQ ID NO: 4, and/or ahistidine at a position corresponding to position 207 of SEQ ID NO: 4;d) a threonine or a valine at a position corresponding to position 15 ofSEQ ID NO: 64, and/or a tyrosine or a valine at a position correspondingto position 203 of SEQ ID NO: 64; e) a leucine at a positioncorresponding to position 199 of SEQ ID NO: 67, and/or a histidine at aposition corresponding to position 200 of SEQ ID NO: 67; and/or f) aleucine at a position corresponding to position 199 of SEQ ID NO: 68, ahistidine at a position corresponding to position 200 of SEQ ID NO: 68,an asparagine at a position corresponding to position 201 of SEQ ID NO:68, and/or a histidine at a position corresponding to position 202 ofSEQ ID NO:
 68. 27. The polypeptide of any one of the preceding claims,wherein the variant IgG Fc polypeptide comprises: a) a threonine atposition 21 of SEQ ID NO: 1, a leucine at position 23 of SEQ ID NO: 1,an alanine at position 25 of SEQ ID NO: 1, a glycine at position 80 ofSEQ ID NO: 1, an alanine at position 205 of SEQ ID NO: 1, and/or ahistidine at position 207 of SEQ ID NO: 1; b) a threonine at position 21of SEQ ID NO: 3, a leucine at position 23 of SEQ ID NO: 3, and/or anisoleucine at position 24 of SEQ ID NO: 3; c) a threonine at a position21 of SEQ ID NO: 4, a leucine at position 23 of SEQ ID NO: 4, an alanineat position 25 of SEQ ID NO: 4, a glycine at position 80 of SEQ ID NO:4, and/or a histidine at position 207 of SEQ ID NO: 4; d) a threonine ora valine at position 15 of SEQ ID NO: 64, and/or a tyrosine or a valineat position 203 of SEQ ID NO: 64; e) a leucine at position 199 of SEQ IDNO: 67, and/or a histidine at position 200 of SEQ ID NO: 67; and/or f) aleucine at position 199 of SEQ ID NO: 68, a histidine at position 200 ofSEQ ID NO: 68, an asparagine at position 201 of SEQ ID NO: 68, and/or ahistidine at position 202 of SEQ ID NO:
 68. 28. The polypeptide of anyone of the preceding claims, wherein the variant IgG Fc polypeptidecomprises an amino acid sequence of: a) SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 84;or b) SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, or SEQID NO:
 76. 29. A polypeptide comprising an amino sequence of SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO: 84, SEQ ID NO: 19, SEQ ID NO: 71, SEQ ID NO: 72, SEQ IDNO: 75, or SEQ ID NO:
 76. 30. The polypeptide of any one of thepreceding claims, wherein the variant IgG Fc polypeptide comprises: a)an amino acid substitution at a position corresponding to position 93 ofSEQ ID NO: 2, or an amino acid substitution at a position correspondingto position 93 of SEQ ID NO: 3; b) an amino acid substitution at aposition corresponding to position 87 of SEQ ID NO: 63, an amino acidsubstitution at a position corresponding to position 87 of SEQ ID NO:65, an amino acid substitution at a position corresponding to position87 of SEQ ID NO: 66, or an amino acid substitution at a positioncorresponding to position 87 of SEQ ID NO: 69; or c) an amino acidsubstitution at a position corresponding to position 198 of SEQ ID NO:80, or an amino acid substitution at a position corresponding toposition 198 of SEQ ID NO:
 81. 31. The polypeptide of any one of thepreceding claims, wherein the variant IgG Fc polypeptide comprises: a)an amino acid substitution at position 93 of SEQ ID NO: 2, or an aminoacid substitution at position 93 of SEQ ID NO: 3; b) an amino acidsubstitution at position 87 of SEQ ID NO: 63, an amino acid substitutionat position 87 of SEQ ID NO: 65, an amino acid substitution at position87 of SEQ ID NO: 66, or an amino acid substitution at position 87 of SEQID NO: 69; or c) an amino acid substitution at position 198 of SEQ IDNO: 80, or an amino acid substitution at position 198 of SEQ ID NO: 81.32. The polypeptide of any one of the preceding claims, wherein thevariant IgG Fc polypeptide comprises: a) an arginine at a positioncorresponding to position 93 of SEQ ID NO: 2, or an arginine at aposition corresponding to position 93 of SEQ ID NO: 3; b) a serine at aposition corresponding to position 87 of SEQ ID NO: 63, a serinesubstitution at a position corresponding to position 87 of SEQ ID NO:65, a serine at a position corresponding to position 87 of SEQ ID NO:66, or a serine at a position corresponding to position 87 of SEQ ID NO:69; or c) an alanine at a position corresponding to position 198 of SEQID NO: 80, or an alanine at a position corresponding to position 198 ofSEQ ID NO:
 81. 33. The polypeptide of any one of the preceding claims,wherein the variant IgG Fc polypeptide comprises: a) an arginine atposition 93 of SEQ ID NO: 2, or an arginine at position 93 of SEQ ID NO:3; b) a serine at position 87 of SEQ ID NO: 63, a serine at position 87of SEQ ID NO: 65, a serine at position 87 of SEQ ID NO: 66, or a serineat position 87 of SEQ ID NO: 69; or c) an alanine at position 198 of SEQID NO: 80, or alanine at position 198 of SEQ ID NO:
 81. 34. Thepolypeptide of any one of the preceding claims, wherein the variant IgGFc polypeptide comprises the amino acid sequence of: a) SEQ ID NO: 78,SEQ ID NO: 79, or SEQ ID NO: 84; or b) SEQ ID NO: 70, SEQ ID NO: 73, SEQID NO: 74, or SEQ ID NO: 77; or c) SEQ ID NO: 82 or SEQ ID NO:
 83. 35. Apolypeptide comprising an amino sequence of SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO: 84, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ IDNO: 77, SEQ ID NO: 82, or SEQ ID NO:
 83. 36. The polypeptide of any oneof the preceding claims, wherein the variant IgG Fc polypeptidecomprises: a) an amino acid substitution at a position corresponding toposition 5 of SEQ ID NO: 2, an amino acid substitution at a positioncorresponding to position 38 of SEQ ID NO: 2, an amino acid substitutionat a position corresponding to position 39 of SEQ ID NO: 2, an aminoacid substitution at a position corresponding to position 97 of SEQ IDNO: 2, and/or an amino acid substitution at a position corresponding toposition 98 of SEQ ID NO: 2; or b) an amino acid substitution at aposition corresponding to position 5 of SEQ ID NO: 3, an amino acidsubstitution at a position corresponding to position 38 of SEQ ID NO: 3,an amino acid substitution at a position corresponding to position 39 ofSEQ ID NO: 3, an amino acid substitution at a position corresponding toposition 97 of SEQ ID NO: 3, and/or an amino acid substitution at aposition corresponding to position 98 of SEQ ID NO:
 3. 37. Thepolypeptide of any one of the preceding claims, wherein the variant IgGFc polypeptide comprises: a) an amino acid substitution at position 5 ofSEQ ID NO: 2, an amino acid substitution at position 38 of SEQ ID NO: 2,an amino acid substitution at position 39 of SEQ ID NO: 2, an amino acidsubstitution at position 97 of SEQ ID NO: 2, and/or an amino acidsubstitution at position 98 of SEQ ID NO: 2; or b) an amino acidsubstitution at position 5 of SEQ ID NO: 3, an amino acid substitutionat position 38 of SEQ ID NO: 3, an amino acid substitution at position39 of SEQ ID NO: 3, an amino acid substitution at position 97 of SEQ IDNO: 3, and/or an amino acid substitution at position 98 of SEQ ID NO: 3.38. The polypeptide of any one of the preceding claims, wherein thevariant IgG Fc polypeptide comprises: a) a proline at a positioncorresponding to position 5 of SEQ ID NO: 2, a glycine at a positioncorresponding to position 38 of SEQ ID NO: 2, an arginine at a positioncorresponding to position 39 of SEQ ID NO: 2, an isoleucine at aposition corresponding to position 97 of SEQ ID NO: 2, and/or a glycineat a position corresponding to position 98 of SEQ ID NO: 2; or b) aproline at a position corresponding to position 5 of SEQ ID NO: 3, aglycine at a position corresponding to position 38 of SEQ ID NO: 3, anarginine at a position corresponding to position 39 of SEQ ID NO: 3, anisoleucine at a position corresponding to position 97 of SEQ ID NO: 3,and/or a glycine at a position corresponding to position 98 of SEQ IDNO:
 3. 39. The polypeptide of any one of the preceding claims, whereinthe variant IgG Fc polypeptide comprises: a) a proline at position 5 ofSEQ ID NO: 2, a glycine at position 38 of SEQ ID NO: 2, an arginine atposition 39 of SEQ ID NO: 2, an isoleucine at position 97 of SEQ ID NO:2, and/or a glycine at position 98 of SEQ ID NO: 2; or b) a proline atposition 5 of SEQ ID NO: 3, a glycine at position 38 of SEQ ID NO: 3, anarginine at position 39 of SEQ ID NO: 3, an isoleucine at position 97 ofSEQ ID NO: 3, and/or a glycine at position 98 of SEQ ID NO:
 3. 40. Thepolypeptide of any one of the preceding claims, wherein the variant IgGFc polypeptide comprises an amino acid sequence of: a) SEQ ID NO: 139,SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ IDNO: 144, SEQ ID NO: 145, SEQ ID NO: 146, or SEQ ID NO: 147; or b) SEQ IDNO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152,SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, or SEQ ID NO:
 157. 41. Apolypeptide comprising an amino sequence of SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 154, SEQID NO: 155, SEQ ID NO: 156, or SEQ ID NO:
 157. 42. A polypeptidecomprising a variant IgG Fc polypeptide comprising: a) a tyrosine or atryptophan at a position corresponding to position 138 of SEQ ID NO: 1,a tyrosine or a tryptophan at a position corresponding to position 137of SEQ ID NO: 2, a tyrosine or a tryptophan at a position correspondingto position 137 of SEQ ID NO: 3, or a tyrosine or a tryptophan at aposition corresponding to position 138 of SEQ ID NO: 4; or b) a tyrosineor a tryptophan at a position corresponding to position 154 of SEQ IDNO: 16, a tyrosine or a tryptophan at a position corresponding toposition 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a tyrosine or atryptophan at a position corresponding to position 154 of SEQ ID NO: 81or SEQ ID NO:
 118. 43. The polypeptide of any one of the precedingclaims, wherein the variant IgG Fc polypeptide comprises: a) a tyrosineor a tryptophan at position 138 of SEQ ID NO: 1, a tyrosine or atryptophan at position 137 of SEQ ID NO: 2, a tyrosine or a tryptophanat position 137 of SEQ ID NO: 3, or a tyrosine or a tryptophan atposition 138 of SEQ ID NO: 4; or b) a tyrosine or a tryptophan atposition 154 of SEQ ID NO: 16, a tyrosine or a tryptophan at position154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a tyrosine or a tryptophan ata position corresponding to position 154 of SEQ ID NO: 81 or SEQ ID NO:118.
 44. The polypeptide of any one of the preceding claims, wherein thevariant IgG Fc polypeptide comprises an amino acid sequence of SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 109, SEQID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO:121, or SEQ ID NO:
 123. 45. A polypeptide comprising an amino acidsequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ IDNO: 119, SEQ ID NO: 121, or SEQ ID NO:
 123. 46. A contiguous polypeptidecomprising the polypeptide of any one of the preceding claims and aglucagon-like peptide-1 (GLP1) polypeptide.
 47. A contiguous polypeptidecomprising the polypeptide of any one of the preceding claims and aglucagon polypeptide.
 48. A polypeptide comprising a variant IgG Fcpolypeptide comprising: a) a serine at a position corresponding toposition 138 of SEQ ID NO: 1, a serine at a position corresponding toposition 137 of SEQ ID NO: 2, a serine at a position corresponding toposition 137 of SEQ ID NO: 3, a serine at a position corresponding toposition 138 of SEQ ID NO: 4, a serine at a position corresponding toposition 154 of SEQ ID NO: 16, a serine at a position corresponding toposition 154 of SEQ ID NO: 80 or SEQ ID NO: 117, or a serine at aposition corresponding to position 154 of SEQ ID NO: 81 or SEQ ID NO:118; b) an alanine at a position corresponding to position 140 of SEQ IDNO: 1, an alanine at a position corresponding to position 139 of SEQ IDNO: 2, an alanine at a position corresponding to position 139 of SEQ IDNO: 3, an alanine at a position corresponding to position 140 of SEQ IDNO: 4, an alanine at a position corresponding to position 156 of SEQ IDNO: 16, an alanine at a position corresponding to position 156 of SEQ IDNO: 80 or SEQ ID NO: 117, or an alanine at a position corresponding toposition 156 of SEQ ID NO: 81 or SEQ ID NO: 118; and/or c) a threonineat a position corresponding to position 181 of SEQ ID NO: 1, a threonineat a position corresponding to position 180 of SEQ ID NO: 2, a threonineat a position corresponding to position 180 of SEQ ID NO: 3, a threonineat a position corresponding to position 181 of SEQ ID NO: 4, a threonineat a position corresponding to position 197 of SEQ ID NO: 16, athreonine at a position corresponding to position 197 of SEQ ID NO: 80or SEQ ID NO: 117, or a threonine at a position corresponding toposition 197 of SEQ ID NO: 81 or SEQ ID NO:
 118. 49. The polypeptide ofany one of the preceding claims, wherein the variant IgG Fc polypeptidecomprises: a) a serine at position 138 of SEQ ID NO: 1, a serine atposition 137 of SEQ ID NO: 2, a serine at position 137 of SEQ ID NO: 3,a serine at position 138 of SEQ ID NO: 4, a serine at position 154 ofSEQ ID NO: 16, a serine at position 154 of SEQ ID NO: 80 or SEQ ID NO:117, or a serine at position 154 of SEQ ID NO: 81 or SEQ ID NO: 118; b)an alanine at position 140 of SEQ ID NO: 1, an alanine at position 139of SEQ ID NO: 2, an alanine at position 139 of SEQ ID NO: 3, an alanineat position 140 of SEQ ID NO: 4, an alanine at position 156 of SEQ IDNO: 16, an alanine at position 156 of SEQ ID NO: 80 or SEQ ID NO: 117,or an alanine at position 156 of SEQ ID NO: 81 or SEQ ID NO: 118;and/or; c) a threonine at position 181 of SEQ ID NO: 1, a threonine atposition 181 of SEQ ID NO: 2, a threonine at position 181 of SEQ ID NO:3, a threonine at position 181 of SEQ ID NO: 4, a threonine at position197 of SEQ ID NO: 16, a threonine at position 197 of SEQ ID NO: 80 orSEQ ID NO: 117, or a threonine at position 197 of SEQ ID NO: 81 or SEQID NO:
 118. 50. The polypeptide of any one of the preceding claims,wherein the variant IgG Fc polypeptide comprises an amino acid sequenceof SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 120, SEQID NO: 122, or SEQ ID NO:
 124. 51. A polypeptide comprising an aminoacid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQID NO: 120, SEQ ID NO: 122, or SEQ ID NO:
 124. 52. The polypeptide ofany one of the preceding claims, wherein the polypeptide isglycoslylated.
 53. The polypeptide of any one of claims 1 to 51, whereinthe polypeptide is aglycosylated.
 54. A contiguous polypeptidecomprising the polypeptide of any one of claims 48 to 53 and aglucagon-like peptide-1 (GLP1) polypeptide.
 55. A contiguous polypeptidecomprising the polypeptide of any one of claims 48 to 53 and a glucagonpolypeptide.
 56. A heterodimeric protein comprising the contiguouspolypeptide of claim 46 and the contiguous polypeptide of claim
 54. 57.A heterodimeric protein comprising the contiguous polypeptide of claim47 and the contiguous polypeptide of claim
 55. 58. The contiguouspolypeptide or heterodimeric protein of any one of claims 46, 47, or 54to 57, wherein the GLP1 polypeptide is a wild-type GLP1 polypeptide,optionally comprising the amino acid sequence of SEQ ID NO:
 85. 59. Thecontiguous polypeptide or heterodimeric protein of any one of claims 46,47, or 54 to 58, wherein the GLP1 polypeptide is a variant GLP1polypeptide.
 60. The contiguous polypeptide or heterodimeric protein ofany one of claims 46, 47, or 54 to 59, wherein the GLP1 polypeptidecomprises the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 87, SEQID NO: 98, or SEQ ID NO:
 99. 61. The contiguous polypeptide orheterodimeric protein of any one of claims 46, 47, or 54 to 60, whereinthe glucagon polypeptide is a wild-type glucagon polypeptide, optionallycomprising the amino acid sequence of SEQ ID NO:
 21. 62. The contiguouspolypeptide or heterodimeric protein of any one of claims 46, 47, or 54to 61, wherein the glucagon polypeptide is a variant glucagonpolypeptide.
 63. A heterodimeric protein comprising: i) a first variantcanine IgG Fc polypeptide comprising at least one amino acidmodification relative to a first wild-type canine IgG Fc polypeptide anda second variant canine IgG Fc polypeptide comprising at least one aminoacid modification relative to a second wild-type canine IgG Fcpolypeptide; or ii) a first variant feline IgG Fc polypeptide comprisingat least one amino acid modification relative to a first wild-typefeline IgG Fc polypeptide and a second variant feline IgG Fc polypeptidecomprising at least one amino acid modification relative to a secondwild-type feline IgG Fc polypeptide, wherein: a) the first variantcanine IgG Fc polypeptide comprises an amino acid substitution at aposition corresponding to position 138 of SEQ ID NO: 1, position 137 ofSEQ ID NO: 2, position 137 of SEQ ID NO: 3, or position 138 of SEQ IDNO: 4; b) the second variant canine IgG Fc polypeptide comprises anamino acid substitution at a position corresponding to position 138,position 140, and/or position 181 of SEQ ID NO: 1, position 137,position 139, and/or position 180 of SEQ ID NO: 2, position 137,position 139, and/or position 180 of SEQ ID NO: 3, or position 138,position 140, and/or position 181 of SEQ ID NO: 4; c) the first variantfeline IgG Fc polypeptide comprises an amino acid substitution at aposition corresponding to position 154 of SEQ ID NO: 6, of SEQ ID NO:80, of SEQ ID NO: 81, of SEQ ID NO: 117, or of SEQ ID NO: 118; and/or d)the second variant feline IgG Fc polypeptide comprises an amino acidsubstitution at a position corresponding to position 154, position 156,and/or position 197 of SEQ ID NO: 6, of SEQ ID NO: 80, of SEQ ID NO: 81,of SEQ ID NO: 117, or of SEQ ID NO:
 118. 64. The heterodimeric proteinof claim 63, wherein the first wild-type canine IgG Fc polypeptide andthe second wild-type canine IgG Fc polypeptide are from the same IgGsubtype and/or the first wild-type feline IgG Fc polypeptide and thesecond wild-type feline IgG Fc polypeptide are from the same IgGsubtype.
 65. The heterodimeric protein of claim 63, wherein the firstwild-type canine IgG Fc polypeptide and the second wild-type canine IgGFc polypeptide are from a different IgG subtype and/or the firstwild-type feline IgG Fc polypeptide and the second wild-type feline IgGFc polypeptide are from the same IgG subtype.
 66. The heterodimericprotein of any one of claims 63 to 65, wherein: a) the first variantcanine IgG Fc polypeptide comprises a tyrosine or tryptophan at aposition corresponding to position 138 of SEQ ID NO: 1, position 137 ofSEQ ID NO: 2, position 137 of SEQ ID NO: 3, or position 138 of SEQ IDNO: 4; and/or b) the first variant feline IgG Fc polypeptide comprises atryptophan at a position corresponding to position 154 of SEQ ID NO: 6,of SEQ ID NO: 80, of SEQ ID NO: 81, of SEQ ID NO: 117, or of SEQ ID NO:118.
 67. The heterodimeric protein of any one of claims 63 to 66,wherein: a) the second variant canine IgG Fc polypeptide comprises aserine at a position corresponding to position 138, an alanine at aposition corresponding to position 140, and/or a threonine at a positioncorresponding to position 181 of SEQ ID NO: 1, a serine at a positioncorresponding to position 137, an alanine at a position corresponding toposition 139, and/or a threonine at a position corresponding to position180 of SEQ ID NO: 2, a serine at a position corresponding to position137, an alanine at a position corresponding to position 139, and/or athreonine at a position corresponding to position 180 of SEQ ID NO: 3,and/or a serine at a position corresponding to position 138, an alanineat a position corresponding to position 140, and/or a threonine at aposition corresponding to position 181 of SEQ ID NO: 4; and/or b) thesecond variant feline IgG Fc polypeptide comprises a serine at aposition corresponding to position 154, an alanine at a positioncorresponding to position 156, and/or a threonine at a positioncorresponding to position 197 of SEQ ID NO: 6, of SEQ ID NO: 80, of SEQID NO: 81, of SEQ ID NO: 117, or of SEQ ID NO:
 118. 68. Theheterodimeric protein of any one of claims 63 to 67, wherein: a) thefirst variant canine IgG Fc polypeptide comprises an amino acid sequenceof SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:109, SEQ ID NO: 111, SEQ ID NO: 113, or SEQ ID NO: 115; and/or b) thefirst variant feline IgG Fc polypeptide comprises an amino acid sequenceof SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO:
 123. 69. Theheterodimeric protein of any one of claims 63 to 68, wherein: a) thesecond variant canine IgG Fc polypeptide comprises an amino acidsequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, or SEQ ID NO: 116;and/or b) the second variant feline IgG Fc polypeptide comprises anamino acid sequence of SEQ ID NO: 120, SEQ ID NO: 122, or SEQ ID NO:123.
 70. The polypeptide, the contiguous polypeptide, or theheterodimeric protein of any one of claims 42 to 69, wherein the variantIgG Fc polypeptide, the first variant IgG Fc polypeptide, and/or thesecond variant IgG Fc polypeptide comprises at least one additionalamino acid modification relative to a wild-type IgG Fc polypeptide andhas increased binding affinity to Protein A relative to the wild-typeIgG Fc polypeptide.
 71. The polypeptide, the contiguous polypeptide, orthe heterodimeric protein of any one of claims 42 to 70, wherein thevariant IgG Fc polypeptide, the first variant IgG Fc polypeptide, and/orthe second variant IgG Fc polypeptide comprises: a) an amino acidsubstitution at a position corresponding to position 21 of SEQ ID NO: 1,an amino acid substitution at a position corresponding to position 23 ofSEQ ID NO: 1, an amino acid substitution at a position corresponding toposition 25 of SEQ ID NO: 1, an amino acid substitution at a positioncorresponding to position 80 of SEQ ID NO: 1, an amino acid substitutionat a position corresponding to position 205 of SEQ ID NO: 1, and/or anamino acid substitution at a position corresponding to position 207 ofSEQ ID NO: 1; b) an amino acid substitution at a position correspondingto position 21 of SEQ ID NO: 3, an amino acid substitution at a positioncorresponding to position 23 of SEQ ID NO: 3, and/or an amino acidsubstitution at a position corresponding to position 24 of SEQ ID NO: 3;or c) an amino acid substitution at a position corresponding to position21 of SEQ ID NO: 4, an amino acid substitution at a positioncorresponding to position 23 of SEQ ID NO: 4, an amino acid substitutionat a position corresponding to position 25 of SEQ ID NO: 4, an aminoacid substitution at a position corresponding to position 80 of SEQ IDNO: 4, and/or an amino acid substitution at a position corresponding toposition 207 of SEQ ID NO:
 4. 72. The polypeptide, the contiguouspolypeptide, or the heterodimeric protein of any one of claims 42 to 71,wherein the variant IgG Fc polypeptide, the first variant IgG Fcpolypeptide, and/or the second variant IgG Fc polypeptide comprises: a)an amino acid substitution at position 21 of SEQ ID NO: 1, an amino acidsubstitution at position 23 of SEQ ID NO: 1, an amino acid substitutionat position 25 of SEQ ID NO: 1, an amino acid substitution at position80 of SEQ ID NO: 1, an amino acid substitution at position 205 of SEQ IDNO: 1, and/or an amino acid substitution at position 207 of SEQ ID NO:1; b) an amino acid substitution at position 21 of SEQ ID NO: 3, anamino acid substitution at position 23 of SEQ ID NO: 3, and/or an aminoacid substitution at position 24 of SEQ ID NO: 3; or c) an amino acidsubstitution at position 21 of SEQ ID NO: 4, an amino acid substitutionat position 23 of SEQ ID NO: 4, an amino acid substitution at position25 of SEQ ID NO: 4, an amino acid substitution at position 80 of SEQ IDNO: 4, and/or an amino acid substitution at position 207 of SEQ ID NO:4.
 73. The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of claims 42 to 72, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises: a) a threonine at a positioncorresponding to position 21 of SEQ ID NO: 1, a leucine at a positioncorresponding to position 23 of SEQ ID NO: 1, an alanine at a positioncorresponding to position 25 of SEQ ID NO: 1, a glycine at a positioncorresponding to position 80 of SEQ ID NO: 1, an alanine at a positioncorresponding to position 205 of SEQ ID NO: 1, and/or a histidine at aposition corresponding to position 207 of SEQ ID NO: 1; b) a threonineat a position corresponding to position 21 of SEQ ID NO: 3, a leucine ata position corresponding to position 23 of SEQ ID NO: 3, and/or anisoleucine at a position corresponding to position 24 of SEQ ID NO: 3;or c) a threonine at a position corresponding to position 21 of SEQ IDNO: 4, a leucine at a position corresponding to position 23 of SEQ IDNO: 4, an alanine at a position corresponding to position 25 of SEQ IDNO: 4, a glycine at a position corresponding to position 80 of SEQ IDNO: 4, and/or a histidine at a position corresponding to position 207 ofSEQ ID NO:
 4. 74. The polypeptide, the contiguous polypeptide, or theheterodimeric protein of any one of claims 42 to 73, wherein the variantIgG Fc polypeptide, the first variant IgG Fc polypeptide, and/or thesecond variant IgG Fc polypeptide comprises: a) a threonine at position21 of SEQ ID NO: 1, a leucine at position 23 of SEQ ID NO: 1, an alanineat position 25 of SEQ ID NO: 1, a glycine at position 80 of SEQ ID NO:1, an alanine at position 205 of SEQ ID NO: 1, and/or a histidine atposition 207 of SEQ ID NO: 1; b) a threonine at position 21 of SEQ IDNO: 3, a leucine at position 23 of SEQ ID NO: 3, and/or an isoleucine atposition 24 of SEQ ID NO: 3; or c) a threonine at position 21 of SEQ IDNO: 4, a leucine at position 23 of SEQ ID NO: 4, an alanine at position25 of SEQ ID NO: 4, a glycine at position 80 of SEQ ID NO: 4, and/or ahistidine at position 207 of SEQ ID NO:
 4. 75. The polypeptide, thecontiguous polypeptide, or the heterodimeric protein of any one ofclaims 42 to 74, wherein the variant IgG Fc polypeptide, the firstvariant IgG Fc polypeptide, and/or the second variant IgG Fc polypeptidecomprises at least one additional amino acid modification relative to awild-type IgG Fc polypeptide and has decreased binding affinity to CD16relative to the wild-type IgG Fc polypeptide.
 76. The polypeptide, thecontiguous polypeptide, or the heterodimeric protein of any one ofclaims 42 to 75, wherein the variant IgG Fc polypeptide, the firstvariant IgG Fc polypeptide, and/or the second variant IgG Fc polypeptidecomprises: a) an amino acid substitution at a position corresponding toposition 5 of SEQ ID NO: 2, an amino acid substitution at a positioncorresponding to position 38 of SEQ ID NO: 2, an amino acid substitutionat a position corresponding to position 39 of SEQ ID NO: 2, an aminoacid substitution at a position corresponding to position 97 of SEQ IDNO: 2, and/or an amino acid substitution at a position corresponding toposition 98 of SEQ ID NO: 2; or b) an amino acid substitution at aposition corresponding to position 5 of SEQ ID NO: 3, an amino acidsubstitution at a position corresponding to position 38 of SEQ ID NO: 3,an amino acid substitution at a position corresponding to position 39 ofSEQ ID NO: 3, an amino acid substitution at a position corresponding toposition 97 of SEQ ID NO: 3, and/or an amino acid substitution at aposition corresponding to position 98 of SEQ ID NO:
 3. 77. Thepolypeptide, the contiguous polypeptide, or the heterodimeric protein ofany one of claims 42 to 76, wherein the variant IgG Fc polypeptide, thefirst variant IgG Fc polypeptide, and/or the second variant IgG Fcpolypeptide comprises: a) an amino acid substitution at position 5 ofSEQ ID NO: 2, an amino acid substitution at position 38 of SEQ ID NO: 2,an amino acid substitution at position 39 of SEQ ID NO: 2, an amino acidsubstitution at position 97 of SEQ ID NO: 2, and/or an amino acidsubstitution at position 98 of SEQ ID NO: 2; or b) an amino acidsubstitution at position 5 of SEQ ID NO: 3, an amino acid substitutionat position 38 of SEQ ID NO: 3, an amino acid substitution at position39 of SEQ ID NO: 3, an amino acid substitution at position 97 of SEQ IDNO: 3, and/or an amino acid substitution at position 98 of SEQ ID NO: 3.78. The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of claims 42 to 77, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises: a) a proline at a positioncorresponding to position 5 of SEQ ID NO: 2, a glycine at a positioncorresponding to position 38 of SEQ ID NO: 2, an arginine at a positioncorresponding to position 39 of SEQ ID NO: 2, an isoleucine at aposition corresponding to position 97 of SEQ ID NO: 2, and/or a glycineat a position corresponding to position 98 of SEQ ID NO: 2; or b) aproline at a position corresponding to position 5 of SEQ ID NO: 3, aglycine at a position corresponding to position 38 of SEQ ID NO: 3, anarginine at a position corresponding to position 39 of SEQ ID NO: 3, anisoleucine at a position corresponding to position 97 of SEQ ID NO: 3,and/or a glycine at a position corresponding to position 98 of SEQ IDNO:
 3. 79. The polypeptide, the contiguous polypeptide, or theheterodimeric protein of any one of claims 42 to 78, wherein the variantIgG Fc polypeptide, the first variant IgG Fc polypeptide, and/or thesecond variant IgG Fc polypeptide comprises: a) a proline at position 5of SEQ ID NO: 2, a glycine at position 38 of SEQ ID NO: 2, an arginineat position 39 of SEQ ID NO: 2, an isoleucine at position 97 of SEQ IDNO: 2, and/or a glycine at position 98 of SEQ ID NO: 2; or b) a prolineat position 5 of SEQ ID NO: 3, a glycine at position 38 of SEQ ID NO: 3,an arginine at position 39 of SEQ ID NO: 3, an isoleucine at position 97of SEQ ID NO: 3, and/or a glycine at position 98 of SEQ ID NO:
 3. 80.The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of claims 42 to 79, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises at least one additional amino acidmodification relative to a wild-type canine IgG Fc polypeptide and hasdecreased binding affinity to C1q relative to the wild-type canine IgGFc polypeptide.
 81. The polypeptide, the contiguous polypeptide, or theheterodimeric protein of any one of claims 42 to 80, wherein the variantIgG Fc polypeptide, the first variant IgG Fc polypeptide, and/or thesecond variant IgG Fc polypeptide comprises an amino acid substitutionat a position corresponding to position 93 of SEQ ID NO: 2, or an aminoacid substitution at a position corresponding to position 93 of SEQ IDNO:
 3. 82. The polypeptide, the contiguous polypeptide, or theheterodimeric protein of any one of claims 42 to 81, wherein the variantIgG Fc polypeptide, the first variant IgG Fc polypeptide, and/or thesecond variant IgG Fc polypeptide comprises an amino acid substitutionat position 93 of SEQ ID NO: 2, or an amino acid substitution atposition 93 of SEQ ID NO:
 3. 83. The polypeptide, the contiguouspolypeptide, or the heterodimeric protein of any one of claims 42 to 82,wherein the variant IgG Fc polypeptide, the first variant IgG Fcpolypeptide, and/or the second variant IgG Fc polypeptide comprises anarginine at a position corresponding to position 93 of SEQ ID NO: 2, oran arginine at a position corresponding to position 93 of SEQ ID NO: 3.84. The polypeptide, the contiguous polypeptide, or the heterodimericprotein of any one of claims 42 to 83, wherein the variant IgG Fcpolypeptide, the first variant IgG Fc polypeptide, and/or the secondvariant IgG Fc polypeptide comprises an arginine at position 93 of SEQID NO: 2, or an arginine at position 93 of SEQ ID NO:
 3. 85. Thepolypeptide, the contiguous polypeptide, or the heterodimeric protein ofany one of claims 1 to 84, wherein the polypeptide is an antibody, anantibody fusion, or a fusion polypeptide.
 86. A contiguous polypeptidecomprising: a) a first glucagon-like peptide-1 (GLP1) polypeptide(GLP1A); b) a first linker (L1); c) an Fc polypeptide (Fc) of acompanion animal species; d) optionally, a second linker (L2); and e)optionally, a second GLP1 polypeptide (GLP1B).
 87. The contiguouspolypeptide of claim 65 comprising:GLP1A-L1-Fc; or  formula (I):Fc-L1-GLP1A.  formula (II):
 88. The contiguous polypeptide of claim 65comprising:GLP1A-L1-Fc-L2-GLP1B.  formula (III):
 89. The contiguous polypeptide ofany one of claims 86 to 88, wherein GLP1B, if present, comprises thesame amino acid sequence as GLP1A.
 90. A contiguous polypeptidecomprising: a) a glucagon-like peptide-1 (GLP1) polypeptide; b) a firstlinker (L1); c) an Fc polypeptide (Fc); d) a second linker (L2); and e)a glucagon polypeptide (Gluc).
 91. The contiguous polypeptide of claim90 comprising:GLP1-L1-Fc-L2-Gluc; or  Formula (IV):Gluc-L1-Fc-L2-GLP1.  Formula (V):
 92. The contiguous polypeptide of anyone of claims 86 to 91, wherein GLP1A, GLP1, and/or GLP1B, if present,comprises a wild-type GLP1 polypeptide.
 93. The contiguous polypeptideof any one of claims 86 to 92, wherein GLP1A, GLP1, and/or GLP1B, ifpresent, comprises a variant GLP1 polypeptide.
 94. The contiguouspolypeptide of any one of claims 86 to 93, wherein GLP1A, GLP1, and/orGLP1B, if present, comprises an amino acid sequence of SEQ ID NO: 85,SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 98, or SEQ ID NO:
 99. 95. Thecontiguous polypeptide of any one of claims 86 to 94, wherein theglucagon polypeptide comprises a wild-type glucagon polypeptide,optionally comprising the amino acid sequence of SEQ ID NO:
 21. 96. Thecontiguous polypeptide of any one of claims 86 to 95, wherein theglucagon polypeptide is a variant glucagon polypeptide.
 97. Thecontiguous polypeptide of any one of claims 86 to 96, wherein the Fcpolypeptide is a human IgG Fc.
 98. The contiguous polypeptide of any oneof claims 86 to 97, wherein the Fc polypeptide is a human IgG1 Fc, IgG2Fc, IgG3 Fc, or IgG4 Fc.
 99. The contiguous polypeptide of any one ofclaims 86 to 98, wherein the Fc polypeptide is an Fc of a companionanimal species.
 100. The contiguous polypeptide of any one of claim 86to 97 or 99, wherein the Fc polypeptide comprises: a) a canine IgG-A Fc,IgG-B Fc, IgG-C Fc, or IgG-D Fc; b) an equine IgG1 Fc, IgG2 Fc, IgG3 Fc,IgG4 Fc, IgG5 Fc, IgG6 Fc, or IgG7 Fc; or c) a feline IgG1a Fc, IgG1bFc, or IgG2 Fc.
 101. The contiguous polypeptide of any one of claims 86to 100, wherein the Fc polypeptide is a wild-type IgG Fc polypeptide.102. The contiguous polypeptide of any one of claims 86 to 100, whereinthe Fc polypeptide is a variant IgG Fc polypeptide.
 103. The contiguouspolypeptide of any one of claims 86 to 102, wherein the Fc polypeptidecomprises the polypeptide, the contiguous polypeptide, or theheterodimeric protein of any one of claims 1 to
 84. 104. The contiguouspolypeptide of any one of claims 85 to 102, wherein the contiguouspolypeptide has a longer serum half-life than a wild-type GLP1polypeptide.
 105. The contiguous polypeptide of any one of claims 86 to104, wherein L1 and L2, if present, each independently is a flexiblelinker.
 106. The contiguous polypeptide of any one of claims 86 to 105,wherein the amino acid sequence of L1 and L2, if present, eachindependently comprises 100%, at least 95%, at least 90%, at least 85%serine and/or glycine amino acid residues.
 107. The contiguouspolypeptide of any one of claims 86 to 106, wherein the contiguouspolypeptide comprises an extension at its C-terminus.
 108. Thecontiguous polypeptide of any one of claims 86 to 107, wherein thecontiguous polypeptide comprises a glycine residue, two glycineresidues, three glycine residues, four glycine residues, five glycineresidues, six glycine residues, seven glycine residues, eight glycineresidues, or greater than eight glycine residues at its C-terminus. 109.The contiguous polypeptide of any one of claims 86 to 108, wherein thecontiguous polypeptide comprises an amino acid sequence of SEQ ID NO:88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ IDNO: 93, SEQ ID NO: 94, or SEQ ID NO: 95 at its C-terminus.
 110. Thecontiguous polypeptide of any one of claims 86 to 109, wherein thecontiguous polypeptide comprises: a) the amino acid sequence of SEQ IDNO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32;SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO:37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ IDNO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQID NO: 47, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 103, SEQ ID NO: 104,SEQ ID NO: 105, or SEQ ID NO: 106; or b) the amino acid sequence of SEQID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56;SEQ ID NO: 57; SEQ ID NO: 58; or SEQ ID NO:
 59. 111. A polypeptidecomprising an amino acid sequence of SEQ ID NO: 23; SEQ ID NO: 24; SEQID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29;SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO:34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ IDNO: 39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQID NO: 44; SEQ ID NO: 45; SEQ ID NO: 46; SEQ ID NO: 47, SEQ ID NO: 96,SEQ ID NO: 97, SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO:55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ IDNO: 103; SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO:
 106. 112. Thepolypeptide, the heterodimeric protein, or the contiguous polypeptide ofany one of the preceding claims, wherein the at least one amino acidmodification or substitution comprises an amino acid substitution withan amino acid derivative.
 113. An isolated nucleic acid encoding thepolypeptide, the heterodimeric protein, or the contiguous polypeptide ofany one of the preceding claims.
 114. A host cell comprising the nucleicacid of claim
 113. 115. A method of producing a polypeptide comprisingculturing the host cell of claim 114 and isolating the polypeptide. 116.A pharmaceutical composition comprising the polypeptide, theheterodimeric protein, or the contiguous polypeptide of any one ofclaims 1 to 112, and a pharmaceutically acceptable carrier.
 117. Amethod of increasing production of cAMP in a cell, the method comprisingexposing the cell to the polypeptide, the heterodimeric protein, thecontiguous polypeptide, or the pharmaceutical composition of any one ofclaim 1 to 112 or 116 under conditions permissive for binding of thepolypeptide, heterodimeric protein, or contiguous polypeptide to GLP1R.118. The method of claim 117, wherein the cell is exposed to thepolypeptide, heterodimeric protein, contiguous polypeptide, or thepharmaceutical composition ex vivo.
 119. The method of claim 117,wherein the cell is exposed to the polypeptide, heterodimeric protein,contiguous polypeptide, or the pharmaceutical composition in vivo. 120.The method of any one of claims 118 to 119, wherein the cell is a humancell, a canine cell, a feline cell, or an equine cell.
 121. A method ofdelivering a polypeptide to a subject comprising administering thepolypeptide, the heterodimeric protein, the contiguous polypeptide, orthe pharmaceutical composition of any one of claim 1 to 112 or 116parenterally.
 122. A method of delivering a polypeptide to a subjectcomprising administering the polypeptide, the heterodimeric protein, thecontiguous polypeptide, or the pharmaceutical composition of any one ofclaim 1 to 112 or 116 by an intramuscular route, an intraperitonealroute, an intracerobrospinal route, a subcutaneous route, anintra-arterial route, an intrasynovial route, an intrathecal route, oran inhalation route.
 123. A method of treating a subject having diabetesor obesity, the method comprising administering to the subject atherapeutically effective amount of the polypeptide, the heterodimericprotein, the contiguous polypeptide, or the pharmaceutical compositionof any one of claim 1 to 112 or
 116. 124. The method of claim 123,comprising administering insulin, a DPP4 inhibitor, a SGLT2 inhibitor, abiguanides sulfonylureas meglitinide derivative, an alpha-glucosidaseinhibitor, a thiazolidinedion (TZD), an amylinomimetic, a bile acidsequestrant, a dopamine agonist.
 125. The method of any one of claims121 to 124, wherein the subject is a human subject.
 126. The method ofany one of claims 121 to 124, wherein the subject is a companion animalspecies.
 127. The method of claim 126, wherein the companion animalspecies is canine, equine, or feline.